Berk2010-Conor: Difference between revisions

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[[Berk2010-Conor (June) | June Entries]]<br>
[[Berk2010-Conor (July) | July Entries]]
==To Do:==
==To Do:==
*Choano Assays
*next stage of 2AB assembly for parts 007 and 013


==[[User:Conor McClune|Conor McClune]] 15:45, 13 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 22:47, 16 October 2010 (EDT)==
===Assembly of iGEM10_006===
===Transposase Assembly===
'''Digestion'''<br>
====Colony PCR analysis====
*iGEM10_005 #3: 4uL DNA, 4uL H2O, 1uL NEB2, 1uL Xhol, 1uL Bam
Colony PCR failed: no bands
*iGEM10_004 #4: 4uL DNA, 4uL H2O, 1uL NEB2, 1uL Xhol, 1uL Bgl
*incubated from 11:19-12:25
<br>
'''Gel Purification'''
*iGEM10_004 #4:
**desired fragment:4841
**other fragment:1475
*iGEM10_005 #3:
**desired fragment:3629
**other fragment:1196
[[image:ConorGel54.jpg|400px]]
<br> I gel purified the desired fragments and zymo purified them. I accidentally used double the water (17uL), so my zymo is a little dilute and I had to modify the ligation procedure<br><br>
'''Ligation'''
*2uL digested iGEM10_004 #4
*2uL digested iGEM10_005 #3
*4uL H20
*1uL T4 ligase buffer with ATP
*1uL T4 Ligase
<br>
'''Transformation'''
*20uL KCM added to 100uL JTK030 comp cells
*all of ligation mixture added to these cells
*sat on ice for 10 minutes
*heat shocked at 42 for 90 seconds
*rescued at 37 on shaker form 2:56-4:02
*plated entire solution on KA plate


==[[User:Conor McClune|Conor McClune]] 14:14, 12 July 2010 (EDT)==
*171+174 (A-D):
===Eco/Bam Transfering Basic parts to correct KA vector===
**lanes 1-4
*used Tim's digested parts and Amy's new digest pMLL6-KA
**expected size: 4615
*I eco/bam digested sbb42 for 1hr and Bjh2245 for 30 min
**good clones:
*created ligase MM:
*171+175 (A-B):
**130uL H20
**lanes 5-6
**20uL T4 ligase buffer
**expected size: 4416
**20uL Eco/Bam digested pMLL6-KA
**good clones:
*added 8.5uL MM to 1uL of each of the following Eco/Bam digested parts:
*170+175 (A-C):
{| {{table}}
**lanes 7-9
| align="center" style="background:#f0f0f0;"|'''Well'''
**expected size: 5265
| align="center" style="background:#f0f0f0;"|'''Ebid'''
**good clones:
| align="center" style="background:#f0f0f0;"|'''Part'''
*170+174 (A-B):
| align="center" style="background:#f0f0f0;"|'''Strain'''
**lanes 10-11
|-
**expected size: 5464
| 1||2||sbb09||R
**good clones:
|-
*170+173 (A-D):
| 2||4||sbb07||R
**lanes 12-15
|-
**expected size: 5471
| 3||5||sbb06||R
**good clones:
|-
| 4||7||sbb04||R
|-
| 5||11||Bjh1906||R
|-
| 6||12||sbb19||R
|-
| 7||19||sbb12||R
|-
| 8||22||sbb10||R
|-
| 9||23||sbb42||R
|-
| 10||24||Bjh2294||R
|-
| 11||na||Bjh2245||R
|-
| 12||7||sbb04||L
|-
| 13||12||sbb19||L
|-
| ||||||
|-
| 15||22||sbb10||L
|-
| 16||23||sbb42||L
|}
*Transformed into the appropriate righty or lefty mc1061 pir+ strain


===Restriction Mapping===
==[[User:Conor McClune|Conor McClune]] 18:11, 13 October 2010 (EDT)==
*digestion master mixes- 4 H2O: 1 NEB2 : 0.5 Enzyme1 : 0.5 Enzyme1


===Transposase assembly===
Conducted the following digestions:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Restr. Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Parent Size'''
| align="center" style="background:#f0f0f0;"|'''Expected Sizes'''
| align="center" style="background:#f0f0f0;"|'''Expected Fragments'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
| align="center" style="background:#f0f0f0;"|'''Gel Purified'''
|-
|-
| 1||sbb10 #1||pMLL6-KA||Eco/Bam||2868, 1035||yes
| 1||163-5a||Xho/Bam||6004||4730, 1270||yes||
|-
|-
| 2||sbb10 #2||pMLL6-KA||Eco/Bam||2868, 1035||yes
| 2||163-5b||Xho/Bam||6004||4730, 1270||yes||yes
|-
|-
| 3||sbb10 #3||pMLL6-KA||Eco/Bam||2868, 1035||yes
| 3||163-10a||Xho/Bam||5150||3880, 1270||no||
|-
|-
| 4||sbb10 #4||pMLL6-KA||Eco/Bam||2868, 1035||yes
| 4||163-10b||Xho/Bam||5150||3880, 1270||no||
|-
|-
| 5||iGEM10_004 #1||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
| 5||163-17b||Xho/Bam||5400||4100, 1270||yes||yes
|-
|-
| 6||iGEM10_004 #2||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
| 6||163-17a||Xho/Bam||5400||4100, 1270||yes||
|-
|-
| 7||iGEM10_004 #4||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
| 7||ladder||||||||||
|-
|-
| 8||iGEM10_005 #6||pMLL8-KC||Eco/Bam||2662, 2163||failed digest (righty)
| 8||163-17b||Xho/Bgl||5400||1475, 3921||yes||
|-
|-
| 9||sbb04 #1||pMLL6-KA||Eco/Bam||2868, 1797||yes
| 9||2142-164a||Xho/Bgl||5090||3408, 1680||yes||yes
|-
|-
| 10||sbb04 #2||pMLL6-KA||Eco/Bam||2868, 1797||yes
| 10||2142-164b||Xho/Bgl||5090||3408, 1680||yes||
|-
|-
| 11||Bjh2294 #1||pMLL6-KA||Eco/Bam||2868, 2516||probably
| 11||2142-165a||Xho/Bgl||5080||3401, 1680||yes||yes
|-
|-
| 12||Bjh2294 #2||pMLL6-KA||Eco/Bam||2868, 2516||probably
| 12||2142-165b||Xho/Bgl||5080||3401, 1680||yes||
|-
|-
| 13||iGEM10_005 #2||pMLL9-CA||Eco/Xho||3227, 1598||yes
| 13||2142-166a||Xho/Bgl||4880||3200, 1680||yes||
|-
|-
| 14||iGEM10_005 #3||pMLL9-CA||Eco/Xho||3227, 1598||yes
| 14||2142-166b||Xho/Bgl||4880||3200, 1680||yes||yes
|-
| 15||iGEM10_005 #4||pMLL9-CA||Eco/Xho||3227, 1598||yes
|-
| 16||iGEM10_005 #8||pMLL9-CA||Eco/Xho||3227, 1598||yes
|}
[[image: ConorGel52.JPG|400px]]
<br>
<br>iGEM10_004 failed to digest because Bam does not digest righty-methylated parts. I am redoing it with an Eco/Xho digest:<br>
[[image: ConorGel53.jpg|400px]]<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Expected Sizes'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_004 #1||pMLL8-KC||Eco/Xho||4850, 1466||too faint to see
|-
| 2||iGEM10_004 #2||pMLL8-KC||Eco/Xho||4850, 1466||too faint to see
|-
| 3||iGEM10_004 #4||pMLL8-KC||Eco/Xho||4850, 1466||looks good
|-
| 4||iGEM10_005 #6||pMLL8-KC||Eco/Xho||4850, 1466||looks good
|}
|}
Gel image:<br>
[[image:ConorGel105.jpg|400px]]


===Eco/Bgl/Bam assembly of iGEM10_006, iGEM10_010, iGEM10_011===
===preparation for plasmid payload cytometry===
'''Digestion'''
====Self lysis test====
*digestion master mixes- 4 H2O: 1 NEB2 : 0.5 Enzyme1 : 0.5 Enzyme1
*started 2:00
*checked at 4:30:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Part'''
| ||1||2||3||6||7
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Colony # or letter'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Desired Frag'''
| align="center" style="background:#f0f0f0;"|'''Other Fragment'''
|-
|-
| iGEM10_005||pMLL8-KC||3||Eco/Bam||2163||2662
| 51||lysed||lysed||lysed||barely lysed||lysed
|-
|-
| sbb10||pMLL6-KA||1||Eco/Bam||1035||2868
| 51||lysed||lysed||lysed||lysed||lysed
|-
| iGEM10_008||pMLL5-CK||d||Eco/Bam||1066||2662
|-
| iGEM10_004||pMLL9-CA||2||Eco/Bgl||6307||9
|-
| iGEM10_009||pMLL4-AC||b||Eco/Bgl||3015||9
|-
| Bjh2294||pMLL6-KA||1||Eco/Bgl||5375||9
|}
|}
*I gel purified all the desired fragments
*note, there was no clearly visible band at 6307 in lane 4, but I cut out a larger area. The above restriction digests of iGEM10_004 suggest that it may have been better to use colony #3 or 4, not #2, as I did.


==[[User:Conor McClune|Conor McClune]] 22:25, 11 July 2010 (EDT)==
===Col PCR results of picking iGEM10_004, iGEM10_005, sbb04 and Bjh2294===
[[image:ConorGel50.jpg|400px]]
*lanes 1-8 : iGEM10_004
**expected size:3771
**wrong size, most lanes seem to match the size of iGEM10_005 instead
*lanes 9-16 : iGEM10_005
**expected size:2354
** none are correct size, though lanes 1,2,4,5,6 match the size of iGEM10_004
*lanes 17-20 : sbb04
**expected size:1988
**all 4 lanes look good
*lanes 21-24 : Bjh2294
**expected size:2707
**all 4 lanes look good
Note: lanes 17-24 and final ladder are somewhat warped due to the plastic object I used to hold the gel
[[image:ConorGel51.jpg|200px]]


I must have switched the plates for iGEM10_005 and iGEM10_004 when I was picking, so I have the correct parts under the wrong labels. From this point one I will switch the iGEM10_005 and iGEM10_004 back.
====preparation of cells====
*scraped choanos and put 1ml in each test tube
*added 40 uL bacteria to each test tube at 2:30
*induced with arabinose fifteen minutes later (at 2:45)


==[[User:Conor McClune|Conor McClune]] 20:15, 10 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 18:31, 12 October 2010 (EDT)==
=== Replenish stcoks of sbb10===
*flow cytometry got no hits, because the choanos were in ASW, so the bacteria did not lyse
*miniprepped all 4 colonies I grew up (though colony #1 looks best)


=== Replenish stocks of sbb04 and Bjh2294 (Self Lysis Device)===
==[[User:Conor McClune|Conor McClune]] 18:31, 11 October 2010 (EDT)==
*picked 4 colonies of each and transferred to KA LB
===preparation for plasmid payload cytometry===
 
====Self lysis test====
===Assembly of iGEM10_004, iGEM10_005 and iGEM10_011===
*started 2:15
*no colonies grew for iGEM10_011, but a few grew up on the plates for iGEM10_004 and iGEM10_005
*checked at 4:15:
 
 
 
==[[User:Conor McClune|Conor McClune]] 15:02, 9 July 2010 (EDT)==
===Retransform Bjh2294 (self lysis device) and sbb04 for stock replentishment===
*used Assembly teams methylated, 1:2 diluted stocks
*transformed 4uL of of each miniprepped DNA into 100uL jtk030 comp cells (w/ 20uL KCM).
*rescued for 1hr at 37*C
*plated entire solutions onto KA plates
 
=== Replenish stocks of sbb10===
*picked 4 colonies from yesterdays transformation and placed in 3uL KA LB
*set up a Col PCR with ca998 and G00101
'''Col PCR results:'''<br>
*expected size:1226
[[image:ConorGel49.jpg|400px]]<br>
''Results:''<br>
Colony one looks great, other colonies show faint lines.
 
=== Assembly of iGEM10_004, iGEM10_005, iGEM10_011===
'''Digestion'''
*made lefty and righty digestion MM
*digested the following parts:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| ||1||2||3||6||7
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''MM'''
| align="center" style="background:#f0f0f0;"|'''Desired Fragment'''
| align="center" style="background:#f0f0f0;"|'''Other Fragment'''
|-
| 1||sbb04||pMLL6-KA||L||3395||1270
|-
| 2||iGEM10_003b||pMLL5-CK||L||2539||1064
|-
| 3||iGEM10_008d||pMLL5-CK||L||2532||1196
|-
|-
| 4||iGEM10_002d||pMLL4-AC||R||1430||1681
| 51||lysed||lysed||lysed||lysed||lysed
|-
|-
| 5||iGEM10_009b||pMLL4-AC||R||1343||1681
| 51||lysed||lysed||lysed||lysed||lysed
|}
|}
*I gel purified the desired bands
*Amy already had gel purified a lefty (xho/bam) digest of Bjh2294


'''Ligation'''
*I did standard ligations to make the following parts:
**sbb04 + iGEM10_002 = iGEM10_004
**iGEM10_003 + Bjh2294 = iGEM10_005
**iGEM10_008 + Bjh2294  = iGEM10_011


'''Transformation'''
====preparation of cells====
*I transformed iGEM10_004 and iGEM10_011 into righty pir+ mc1061
*scraped choanos and put 6ml in each test tube
**these I plated on CA plates after an hour's rescue
*added 240 uL bacteria to each test tube
*I transformed iGEM10_005 into lefty pir+ mc1061
*induced with arabinose fifteen minutes later (at 2:30)
**these I plated on a KC plate after an hour's rescue


==[[User:Conor McClune|Conor McClune]] 14:03, 8 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 19:02, 6 October 2010 (EDT)==
===Assembly of iGEM10_007 and iGEM10_013===
===Plasmid Delivery Assay===
====Restriction Digest of iGEM10_004,iGEM10_005 and iGEM10_011====
''Plasmid Payload Numbers:''
Samples checked: iGEM10_004#7 ,iGEM10_005#1 and iGEM10_011#1 <br>
Well contents:
*12uL miniprepped DNA
*2uL BamHI
*2uL EcoR1
*1.5 NEB2
incubated at 37 for 1hr
'''Gel Results:'''<br>
[[image:ConorGel47.jpg|400px]]<br>
GEM10_004 #7:
*expected sizes:2756,3580
*smeared, but does not look like there are two bands
iGEM10_005 #1:
*expected sizes:2662,2163
*smeared, but it looks like there is an incorrect band around 1kb
iGEM10_011 #1:
*expected sizes:3573,2736
*smeared, second band possibly present, though faint
===Analytical PCR of iGEM10_004,iGEM10_005 and iGEM10_011===
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Number'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part Checked for'''
| align="center" style="background:#f0f0f0;"|'''Antibiotic'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
|-
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| 1||pDYAL eGFP||Amp
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
|-
| align="center" style="background:#f0f0f0;"|'''Acceptable Band?'''
| 2||pDYAL eGFP Swar||Amp
|-
|-
| 1||iGEM10_004 #7||iGEM10_003||ca998||igemTen008||1164||yes, but also a band at 300bp
| 3||Clipit GFP||Amp
|-
|-
| 2||iGEM10_004 #7||Bjh2294||igemTen007||G00101||2607||no
| 4||M cherry||Amp
|-
|-
| 3||iGEM10_005 #1||sbb04||ca998||igemTen004||1888||very faint band
| 5||Tera\'s α-tub GFP jtk159||Amp
|-
|-
| 4||iGEM10_005 #1||iGEM10_002||igemTen003||igemTen006||366||no
| 6||Tera\'s efla GFP jtk160||Amp
|-
|-
| 5||iGEM10_011 #1||iGME10_008||ca998||igemTen020||1157||yes, but also a band at 400bp
| 7||PfuGWP GFP||Amp
|-
|-
| 6||iGEM10_011 #1||Bjh2294||igemTen019||G00101||2607||no
| 8||Tera\'s myo GFP jtk162||Amp
|}
|}
*Well contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
*run on program 3K45
[[image:ConorGel48.jpg|400px]]<br>
''Results'':
*bands in lanes 1,3,5, but not 2,4,6, indicating that none of the parts were assembled correctly. It looks like there is only the lefty half of each part in these plasmids.
====Remake of basic part Sbb10 from 140L stocks====
I have not been able to digest it and the Robot Assembly team has never been able to successfully use it in an assembly, so I am going to re-eco/bam it from a BioE 140L stock.
''Digest:'' Well contents: 4uL H20, 4uL stock sbb10, 1.1uL NEB2, 1uL Eco, 1uL Bam. Digested for 1hr.
<br><br>
''Ligation:'' Ligated into pMLL6 using standard procedure.<br><br>
''Transform:''Transformed ligation mixture into 100uL jtk030 comp cells (w/ 20uL KCM).
*Rescued for 1.5 hour.
*plated entire culture on KA plate
==[[User:Conor McClune|Conor McClune]] 15:08, 7 July 2010 (EDT)==
===2AB assembly of iGEM10_007 and iGEM10_013===
====Failure of iGEM10_010====
Due to all the cotransformations I am getting, I am going to gel purify the digestion fragments
*sbb10:
**digested with Xhol/BamH1
**expected sizes: 2633*, 1026
*iGEM10_009:
**digest with Xhol/BglII
**expected sizes: 1343*, 1681
Gel Results of yesterdays digestions: <br>
[[image:ConorGel44.jpg|400px]]<br>
*I gel purified the desired fragment of iGEM10_009, but it seems sbb10 was not digested well, so I will redo the digestion
====Digestion====
I will be redoing the digestion of sbb10 while also digesting iGEM10_004, iGEM10_005, iGEM10_011 for restriction mapping.<br><br>
'''Sbb10'''
*3uL H20
*8uL (1/2 diluted) miniprepped DNA (lefty methylated)
*1.5uL NEB2 + ATP
*1uL Xhol
*1uL BamH1
[[image:ConorGel46.jpg|400px]]<br>
*the part is still not digested. potential problems:
**bad enzyme?
<br>
'''Restriction Digests'''
*created 24uL Eco/Bam MM
*added 6uL MM to 4uL of iGEM10_004 #7, iGEM10_005 #1, iGEM10_011 #1
Results:<br>
[[image:ConorGel45.jpg|400px]]<br>
iGEM10_004 #7: 2756,3580
*expected sizes:
iGEM10_005 #1:
*expected sizes:2662,2163
iGEM10_011 #1:
*expected sizes:3573,2736
'''Results'''
*unclear, though only one band seems visible. I will rerun the digestion with a higher volume of DNA
==[[User:Conor McClune|Conor McClune]] 17:11, 6 July 2010 (EDT)==
===Manual Assembly of iGEM10_004 ,iGEM10_005 ,iGEM10_010 and iGEM10_011===
'''Picking Colonies'''
*picked 8 colonies of each transformation
*set up Col PCR and swiped cotransformation plate
Col PCR results:<bR>
*note, all the PCR results seem to be wrong, but many are off by the same amount, so perhaps the ladder is off<br>
lanes 1-8:iGEM10_004 - expected size 3771
<br>lanes 10-17: iGEM10_005 - expected size 2354 <br>
[[image:ConorGel41.jpg|400px]]
*correct bands:
**iGEM10_004: lanes 4-7, with 7 being the strongest
***6 is cotransformed
***miniprep: 4,5,7
**iGEM10_005: lanes 1-3, 5-8
***none are cotransformed
***miniprep 1,2,3
<br><br>
lanes 1-8:iGEM10_010 - expected size 1505 <br>
[[image:ConorGel42.jpg|400px]]
*correct bands:
**none - 100% cotransformed
<br><br>
lanes 1-8:iGEM10_011 - expected size 3764 <br>
[[image:ConorGel43.jpg|400px]]
*correct bands:
**lanes 1,3 (neither cotransformed)


===Transformation of Bjh2294 to replenish stock===
*51 and 52 refer to the Self-lysis/Vacuole-buster devices iGEM10_051-052
*yesterday's transformation failed, because there were no colonies
*I will redo the transformation today
Procedure:
*add 50uL H20, 30uL KCN and 2uL DNA to 150uL mc1061 competent cells
*keep on ice for 20 min
*heat shock for 90 sec at 42*C
*mixed with 200uL 2YT and rescue for 1hr
*plate on KA plate


==[[User:Conor McClune|Conor McClune]] 18:14, 5 July 2010 (EDT)==
===Procedure===
===2AB Assembly of Parts iGEM10_007 and iGEM10_013===
''Choanos''
===Digestion for Restriction Mapping and Assembly===
*aspirated two plates (15ml each) of choanos in ASW, resuspended each in 5mL of choano media
*made 7uL lefty and righty digestion master mix
**note: the choano media we planned on using was contaminated, so I had to use an older one (one that seemed to be lacking something because choanos have not grown very well in it)
*added made double the digestion solution for parts we constructed, so I would be able to run a restriction mapping gel with half:
*scraped choanos, mixed the two plates (to get 10mL), aliquoted 500uL into each of 12 mini slide wells
*added 20uL of bacteria to the wells (10:15am):
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| 1+51||2+51||3+51||6+51||7+51||lifeact iGEM10_171 +51(color control)
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''MM'''
| align="center" style="background:#f0f0f0;"|'''Volume MM'''
|-
| 1||sbb04||7-H1||L||7
|-
| 2||iGEM10_003b||||L||14
|-
| 3||sbb10||22-A3||L||7
|-
|-
| 4||iGEM10_008d||||L||14
| 1+52||2+52||3+52||6+52||7+52||white bacteria
|-
| 5||iGEM10_002d||||R||14
|-
| 6||Bjh2294||24-G10||R||7
|-
| 7||iGEM10_009b||||R||14
|-
| 8||Bjh2294||24-G10||R||7
|}
|}
*incubated wells at 37 for 1hr
*mistake:added 0.5uL ATC to each well at 10:30am, instead of arabinose (SLD is under Pbad not Ptet)
*zymo purified each of the wells
*correction: added 5uL arabinose to each well at 11:00am
*used one uL of each purified digest for ligation
 
**combined wells:
''Self lysis test''
***1 with 5 (to make iGEM10_004)
**made two 2mL aliquots of each of the plasmid bacteria cultures listed above
***2 with 6(to make iGEM10_005)
**added 2uL ATC (mistake) and 20uL of arabinose to one of each of the aliquots of each culture
***3 with 7(to make iGEM10_010)
 
***4 with 8(to make iGEM10_011)
===Results===
<br>
''Self Lysis Test''
'''Restriction Digest'''
*after 5 hours, all 10 plasmid+SLD/VB cultures with arabinose had lysed.
*digested with lefty MM (Xhol/BamHI)
**iGEM10_003b - expected sizes:2671,1064
**iGEM10_008d - expected sizes:2664,1064
*digested with righty MM (Xhol/BglII)
**iGEM10_002d - expected sizes:1681, 1430
**iGEM10_009b - expected sizes:1681, 1343
[[image:ConorGel40.jpg|400px]]<br>
''Results'': The strongest bands fit the desired bands. It looks like all these parts are good, though there are some other junk bands.


===Retransformation of Bjh2296===
==[[User:Conor McClune|Conor McClune]] 22:30, 26 September 2010 (EDT)==
*our first stock of Bjh2296 was getting low, so I retransformed it in mc1061
===Eco/Bam transfer of iGEM10_171 (A) into Bca1256===
*note: 2YT used for rescue may be contaminated
*transformed product into JTK159  and plated on spec
*plated on KA plate
===Re-Assembly of iGEM10_170===
*gel purified my previous digestions of iGEM10_159 and Bjh1906
*ligated 4uL of each with 1uL t4 buffer and 1uL t4 ligase
*transformed into JTK159 and plated on CA


==[[User:Conor McClune|Conor McClune]] 14:45, 4 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 16:34, 23 September 2010 (EDT)==
===2AB construction of iGEM10_007 and iGEM10_013===
===LifeAct Assembly===
'''Results from yesterdays Col PCR of iGEM10_004 and iGEM10_011'''
Of the colonies if iGEM10_170 and iGEM10_171 picked yesterday, colonies 5,8,12 and 15 were cotransformed
*iGEM10_004 - expected size: 3771
[[image:ConorGel38.jpg|400px]]


*iGEM10_011 - expected size: 3764
Miniprepped: Colonies 1,2,3,7 (iGEM10_170) and colonies 10,11,18, 20 (iGEM10_171) because they were the reddest
[[image:ConorGel39.jpg|400px]]
*renamed these clones iGEM10_170 A-D and iGEM10_171 A-D, respectively
**iGEM10_171A sequenced well, with one mutation in the promoter
**all of iGEM10_170 A-D are missing the terminator


'''Results:'''<br>
==[[User:Conor McClune|Conor McClune]] 19:55, 22 September 2010 (EDT)==
None of the col PCR fragments are the correct size for either of the parts.
===LifeAct Assembly===
<br>
There is growth on the plates I made yesterday for iGEM10_170-171, though the Red/green colonies on iGEM10_171 are very small
Next Steps:
*do restriction digest to make sure the parts I am combining are correct
*redo 2AB assembly


==[[User:Conor McClune|Conor McClune]] 16:41, 3 July 2010 (EDT)==
I picked 8 colonies for iGEM10_170 and 12 from iGEM10_171 (only the last 4 may be green AND red).
===2AB construction of iGEM10_007 and iGEM10_013===
'''Picking colonies'''<br>
*picked 8 colonies each for iGEM10_004 and iGEM10_011
*swiped each colongy on a AMp/Cam/Kan plate to check for cotransformation
*ran 4K col pcr


==[[User:Conor McClune|Conor McClune]] 20:13, 2 July 2010 (EDT)==
Ran a colony PCR, but it did not show much because the addition of the terminator is not visible on the gel. The gel for the last colonies
===2AB construction of iGEM10_007 and iGEM10_013===
*problem: yesterday I plated the two lefty cultures (containing parts iGEM10_004 and iGEM10_011) on KA plates rather than CA plates. Luckily I had saved the digestions from yesterday, so I religated  and retransformed
<br>
'''Picking Colonies'''
*picked 4 colonies each from the plates containing iGEM10_005 and iGEM10_010
*swiped each colony on a cotransformation plate (Amp) after dipping in col PCR solution and 2uL KC media
''Col PCR''
*lanes 1-4: iGEM10_010 -expected size is 1500
*lanes 5-8: iGEM10_005 -expected size is 2350
[[image:ConorGel36.jpg|400px]]
*none of the colonies for either part are the right size. Both colonies need to be repicked<br>
'''Repicking Colonies'''
*picked 8 more colonies each of iGEM10_010 and iGEM10_005
*ran a col PCR on COL4K
*swiped each colony on an Amp plate to check for cotransformation
'''Results'''
*all 8 colonies of iGEM10_010 are cotransformed
*Gel results of col PCR for iGEM10_005 (expected size 2350):
[[image:ConorGel37.jpg|400px]]


===Choano Assays===
[[image:ConorGel104.jpg|200px]]
*on Tuesday(3 days ago), I split choanos 1:15 into choano media under the following conditions:
*lanes 1-8: iGEM10_170
**25*C(control)
**all but colony 8 look good
**37*C incubator
*lanes 9-13: iGEM10_171
**37*C with shaking
**all look good, except that col PCR failed for colony 12
**1mM NH4
**10mM NH4
*results were unconclusive, because even in the control choano density was too low for counts to be reliable, and florescence was too dim to see


==[[User:Conor McClune|Conor McClune]] 13:34, 1 July 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 18:18, 21 September 2010 (EDT)==
===2AB assembly for iGEM10_007 and iGEM10_013===
===Restriction digest (Eco/Bam)of iGEM10_159-160===
{| {{table}}
*used iGEM10_159C
| align="center" style="background:#f0f0f0;"|''''''
*expected band sizes: 2660, 1580
| align="center" style="background:#f0f0f0;"|'''Lefty'''
[[image:ConorGel103.jpg|200px]]
| align="center" style="background:#f0f0f0;"|''''''
*the bands look good for both (faintess can be attributed to a bad zymo-forgot to spin out solvent for 90 sec before adding water)
| align="center" style="background:#f0f0f0;"|'''Righty'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Product'''
|-
| ||'''Part'''||'''Version'''||'''Part'''||'''Version'''||
|-
| ''iGEM10_007''||||||||||
|-
| ||sbb04||n/a||iGEM10_002||d||iGEM10_004
|-
| ||iGEM10_003||a||Bjh2294||n/a||iGEM10_005
|-
| ''iGEM10_013''||||||||||
|-
| ||sbb10||n/a||iGEM10_009||b||iGEM10_010
|-
| ||iGEM10_008||d||Bjh2294||n/a||iGEM10_011
|}
'''Digestion'''
*made up 35uL of lefty and righty MM
*mixed 4 uL of sbb04,iGEM10_003b,sbb10,iGEM10_008d each with 7uL lefty MM
*mixed 4 uL of iGEM10_002d,Bjh2294,iGEM10_009,Bjh2294 each with 7uL righty MM
*incubated for 1hr at 37*C
'''Ligation'''
*in each ligation: 6.5uL H2O, 1uL ligation buffer, 0.5uL ligase, 1uL lefty DNA, 1uL righty DNA
*Transformed into 70uL lefty and righty cells (parts 004 and 011 into lefty strain, and parts 005 and 010 into righty strain).
*plated lefty strains on CA and righty strains on KC plates
===Analytical PCR of iGEM10_020===
*Well contents: 33uL Taq/DMSO MM, 1uL template DNA, 1uL each oligo
*program: 4K55
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''reverse oligo'''
| align="center" style="background:#f0f0f0;"|'''best annealing temp'''
| align="center" style="background:#f0f0f0;"|'''parts tested for:'''
| align="center" style="background:#f0f0f0;"|'''expected size'''
|-
| 1||iGEM10_020a||ca56||igemTen022||55||igem10_001 and igem10_017||1800
|-
| 2||iGEM10_020a||igemTen021||G00101||55||igem10_016 and Bjh2294||3500
|-
| 3||iGEM10_020b||ca56||igemTen022||55||igem10_001 and igem10_017||1800
|-
| 4||iGEM10_020b||igemTen021||G00101||55||igem10_016 and Bjh2294||3500
|}
''Gel Results''<br>
[[image:ConorGel35.jpg|400px]]
*iGEM10_020b is complete


==[[User:Conor McClune|Conor McClune]] 20:42, 30 June 2010 (EDT)==
===Assembly of iGEM===
===Tn5 Transposase Assay===
Digested:
Testing to see whether the Transposase if functional.
*Xho/Bam:  iGEM10_159C and iGEM10_160
*Xho/Bgl: Bjh1906<br>
Ligated each of the iGEM parts to left of Bjh1906 to make iGEM10_170-171, respectively
<br>
Transformed into JTK159 cells and plated on CA


Specifically, we varied atc concentration and the time allowed for transposase activity.
==[[User:Conor McClune|Conor McClune]] 19:59, 18 September 2010 (EDT)==
===LifeAct Assembly===
====Redigestion of iGEM10_156A and iGEM10_159D====
(this is necessary because the previous version of
*Both were digested with Xho/Bam for 30 min
*Zymo'd
*Ligation (5:00-5:30):
**iGEM10_156A + Bjh1881
**iGEM10_159D + Bjh1906 (terminator)
**iGEM10_159A + Bjh1906 (terminator)


Inoculated cells drived from another colony off the iGEM10_020A plate, which didn't undergo co-transformation. We can use these for tests tomorrow.  
''Results:'' no growth for the iGEM10_159 + Bjh1906 assemblies, but one red/green colony for iGEM10_156A + Bjh1881 = iGEM10_160
*colony PCR of iGEM10_160:
[[image:ConorGel102.jpg|100px]]
*expected band : 1800bp, the band looks good


*Added 10uL of 20% Arabinose (100X) to 1mL of lefty pir+ cells, transformed with iGEM10_020A (no co-transformation occured) at 1:30pm.
====Remaking iGEM10_161 and iGEM10_162====
*At 2:30pm, will add varying amounts of atc to cells.
(This is necessary because I had mislabelled iGEM10_157-158)<br>
**1uL of 1000X atc diluted 1:10
yesterday:  
**1uL of 1000x atc
*ligated the predigested combinations:
**3uL of 1000x atc
**iGEM10_157 + Bjh1881
*At 3:30pm, will add 1uL of pBca9145-Bca1144DNA, and allow the transposase to do it's thing for varying amounts of time.
**iGEM10_158 + Jtk2541
**3:45pm (15min):Remove 250uL of lysate from each tube and spin down and zymo the supernatant.
today:
**4:00pm (30min): See above
*transformed both ligations into JTK159 strain
**4:15pm (45min): See above
*plated on CK
**4:30pm (1hour): See above (note: less than 250uL was left at this point)
*Transformed each of the 12 zymo purified DNAs into 70uL wild-type mc1061 cells
*incubated for 1 hours
*plated on Amp/Gen plates to grow up overnight
'''Results'''
*no colonies on any of the 12 plates


''Results:'' no growth, failed assembly


Note: Talked to Terry, and he said one thing we definitely need to vary is the amount of time we give for Transposase expression.
==[[User:Conor McClune|Conor McClune]] 18:38, 17 September 2010 (EDT)==
===LifeAct Assembly===
====Restriction digest of iGEM10_159-162  with Eco/Bam====
* expected sizes for all bands: 2660, 1580
*iGEM10_159: lanes 1-4
*iGEM10_160: lanes 5-8
*iGEM10_161: lanes 9-12
*iGEM10_162: lanes 13-16
[[Image:Conor 9-14-10.JPG|400px]]


Potential Problem: Used 2YT-Amp for the tubes colored with green sharpie. Hopefully this won't be a problem, since it's sorta the same thing as immediately plating on Amp.
[[Image:Conor 9-14-10 2.JPG|400px]]


==[[User:Conor McClune|Conor McClune]] 14:36, 29 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 18:38, 14 September 2010 (EDT)==
===Gibson Assembly of iGEM10_007 and iGEM10_013===
===Assembly of LifeAct Payload===
====Restriction Mapping====
'''Potential Error discovered:''' ''plate with Eco/Bam iGEM157 and iGEM10158 were probably mislabelled since 157 was red and 158 was green (they should have been the green and red, respectively). This means that iGEM161-162 are probably just two copies of the same FP, one lifeact and one not.''
In order to save time, I also am running a digest of iGEM10_007 for convenience, and of Bth030 for comparison (because it is about 6kb in length). <br>
Well contents:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''well'''
| align="center" style="background:#f0f0f0;"|'''Contents'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
|-
| 1||iGEM10_013 #14||Eco/Bam||6204
|-
| 2||iGEM10_013 #16||Eco/Bam||6204
|-
| 3||iGEM10_007 #11||Eco/Bam||7060
|-
| 4||iGEM10_007 #13||Eco/Bam||7060
|-
| 5||Bth030||Eco/Bam||5850
|}
====Analytical PCR====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Part Checked'''
| align="center" style="background:#f0f0f0;"|'''fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''rev oligo'''
| align="center" style="background:#f0f0f0;"|'''expected size'''
|-
| 1||iGEM10_013 #14||iGEM10_001||ca998||igemTen002||1435
|-
| 2||iGEM10_013 #14||B10sbb10||igemTen034||igemTen036||1026
|-
| 3||iGEM10_013 #14||iGEM10_009||igemTen035||igemTen018||279
|-
| 4||iGEM10_013 #14||iGEM10_008||igemTen017||igemTen020||1057
|-
| 5||iGEM10_013 #14||Bjh2294  w/ fwd oligo 19||igemTen019||G00101||2607
|-
| 6||iGEM10_013 #16||iGEM10_001||ca998||igemTen002||1435
|-
| 7||iGEM10_013 #16||B10sbb10||igemTen034||igemTen036||1026
|-
| 8||iGEM10_013 #16||iGEM10_009||igemTen035||igemTen018||279
|-
| 9||iGEM10_013 #16||iGEM10_008||igemTen017||igemTen020||1057
|-
| 10||iGEM10_013 #16||Bjh2294  w/ fwd oligo 19||igemTen019||G00101||2607
|}
[[image:ConorGel33.jpg|400px]]


===Repicking and Colony PCR===
==[[User:Conor McClune|Conor McClune]] 22:03, 12 September 2010 (EDT)==
*picked 8 colonies each of iGEM10_007 and iGEM10_013
===Lifeact Payload assembly===
*ran two colony PCRs for each colony using the following oligos:
====Digestion====
**for 007
*Lefty digested: iGEM10_155-158
***ca56 + igemTEN4
*Righty digested: Bjh1881, Jtk2541
***igemTEN3+igemTEN8
*incubated 3:30-4:20
**for 013
***ca56+igemTEN36
***igemTEN35 + igemTEN20
''Gel Results''
*lane organization:  
**lanes 1-16 are eight 007 colonies, odd lanes have primers ca56 + igemTEN4, even lanes have primers igemTEN3+igemTEN8
**lanes 1-16 are eight 013 colonies, odd lanes have primers ca56 + igemTEN36, even lanes have primers igemTEN35+igemTEN20
[[image:ConorGel34.jpg|400px]]
<br>
Results:
There are no distinct bands in any lanes. At this point, I think it best to do another round of 2AB assembly before attempting gibson again for parts iGEM10_007 and iGEM10_013.


==[[User:Conor McClune|Conor McClune]] 18:13, 28 June 2010 (EDT)==
====Ligation====
===Choano Heat/NH4 Assays===
*ligating the following combinations:
*on Friday (3 days ago), I split choanos 1:8 into choano media under the following conditions:
**iGEM10_155 + Jtk2541
**25*C(control)
**iGEM10_156 + Bjh1881
**37*C incubator
**iGEM10_157 + Bjh1881
**37*C with shaking
**iGEM10_158 + Jtk2541
**1mM NH4
**10mM NH4
*results were unconclusive, because even in the control choano density was too low for counts to be reliable, however, here are the counts I did get from PI staining:
**control: 1/4 dead
**1mM NH4: 12/38 dead
**37*C incubator:7/17 dead
* I got a fresh culture of MbFb from the king lab so I can redo this experiment starting tomorrow


===iGEM10_007===
==[[User:Conor McClune|Conor McClune]] 01:09, 10 September 2010 (EDT)==
====Analytical PCR====
===Lifact Assembly===
Well contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
*Eco/Bam'd iGEM10_155-158 into pMLL9 (CA) (for continued assembly) and also into 1256 (Spec) (for assaying in choanos)
*run on 4K45


{| {{table}}
==[[User:Conor McClune|Conor McClune]] 01:28, 9 September 2010 (EDT)==
| align="center" style="background:#f0f0f0;"|'''Well'''
===Lifact Payload Assembly===
| align="center" style="background:#f0f0f0;"|'''Sample'''
*I Eco/Bam digested iGEM10_155-158
| align="center" style="background:#f0f0f0;"|'''Part Checked'''
*Gel purified smaller fragment of each
| align="center" style="background:#f0f0f0;"|'''fwd oligo'''
*meant to eco/bam into pMLL CA but we were out of the digested vector. I will have to digest this tomorrow
| align="center" style="background:#f0f0f0;"|'''rev oligo'''
| align="center" style="background:#f0f0f0;"|'''expected size'''
|-
| 1||iGEM10_007 #11||iGEM10_001||ca998||igemTen002||1435
|-
| 2||iGEM10_007 #11|| (B10sbb04)||igemTen001||igemTen004||1788
|-
| 3||iGEM10_007 #11||iGEM10_002||igemTen003||igemTen006||366
|-
| 4||iGEM10_007 #11||iGEM10_003||igemTen005||igemTen008||1064
|-
| 5||iGEM10_007 #11||Self Lysis Device (Bjh2294)||igemTen007||G00101||2607
|-
| 6||iGEM10_007 #13||iGEM10_001||ca998||igemTen002||1435
|-
| 7||iGEM10_007 #13|| (B10sbb04)||igemTen001||igemTen004||1788
|-
| 8||iGEM10_007 #13||iGEM10_002||igemTen003||igemTen006||366
|-
| 9||iGEM10_007 #13||iGEM10_003||igemTen005||igemTen008||1064
|-
| 10||iGEM10_007 #13||Self Lysis Device (Bjh2294)||igemTen007||G00101||2607
|}


[[image:ConorGel30.jpg|400px]]
==[[User:Conor McClune|Conor McClune]] 18:04, 7 September 2010 (EDT)==
===iGEM10_013===
===Lifact Assembly===
====Analytical PCR====
====Colony PCR results====
Well contents: 33uL Taq MM, 1uL each primer, .5 uL template DNA
*colony # corresponds to lane #
{| {{table}}
*expected sizes:
| align="center" style="background:#f0f0f0;"|'''Well'''
**lanes 1-4 Bca1237 + Bjh2252:1070
| align="center" style="background:#f0f0f0;"|'''Sample'''
**lanes 5-8 Bca1238 + Bjh2252:1070
| align="center" style="background:#f0f0f0;"|'''Part Checked'''
**lanes 9-4 Bca1237 + Bjh2251:1020
| align="center" style="background:#f0f0f0;"|'''fwd oligo'''
**lanes 1-4 Bca1238 + Bjh2251:1020
| align="center" style="background:#f0f0f0;"|'''rev oligo'''
| align="center" style="background:#f0f0f0;"|'''expected size'''
|-
| 1||iGEM10_013 #14||iGEM10_001||ca998||igemTen002||1435
|-
| 2||iGEM10_013 #14||B10sbb10||igemTen034||igemTen036||1026
|-
| 3||iGEM10_013 #14||iGEM10_009||igemTen035||igemTen018||279
|-
| 4||iGEM10_013 #14||iGEM10_008||igemTen017||igemTen020||1057
|-
| 5||iGEM10_013 #16||iGEM10_001||ca998||igemTen002||1435
|-
| 6||iGEM10_013 #16||B10sbb10||igemTen034||igemTen036||1026
|-
| 7||iGEM10_013 #16||iGEM10_009||igemTen035||igemTen018||279
|-
| 8||iGEM10_013 #16||iGEM10_008||igemTen017||igemTen020||1057
|-
| 9||iGEM10_013 #14||Bjh2294  w/ fwd oligo 19||igemTen019||G00101||2607
|-
| 10||iGEM10_013 #16||Bjh2294  w/ fwd oligo 19||igemTen019||G00101||2607
|}
*I placed this on the plate and selected the program, but failed to hit start, so the wells sat overnight at room temp.
*I restarted the program in the morning, but it is doubtful whether it will yield good results.
[[image:ConorGel32.jpg|400px]]


==[[User:Conor McClune|Conor McClune]] 19:19, 27 June 2010 (EDT)==
[[image:ConorGel101.jpg|400px]]
===iGEM10_013===
*note: broken well in lane 8, so contents were put in adjacent well
====Picking colonies/Colony PCR====
Results:
*picked 16 colonies
*all lanes look good
*oligos used for col PCR: ca56 and igemTen020
**expected band size = 2619
''Gel Results''<br>
(These are the same gels, but run for different lengths)<br>
[[image:ConorGel28.jpg|400px]]
[[image:ConorGel29.jpg|400px]]
<br> ''Results''
*no bands in any of the chosen colonies, however it seems from the short run on the gel that at least one of the primers was binding to to colonies #14 and 16, as there is a large amount of small fragments of DNA


===iGEM10_020===
==[[User:Conor McClune|Conor McClune]] 21:06, 5 September 2010 (EDT)==
====Miniprep====
===Lifact Payload Assembly===
Friday's col PCR showed that all the colonies selected for the second col PCR had iGEM10_001 transferred into it. The last 4 lanes appeared to have the strongest bands, so I will choose those colonies to miniprep. I will call colonies represented by lanes 13-16 iGEM10_020 a-d, respectively.
*Ran a gel of Tahoura's PCR of Pcon parts Bca1237 and Bca1238:
**image: [[image:ConorGel100.jpg|100px]]<br>
**cut out 200-300bp range for both parts and small fragment gel purified (eluted with 8.5uL H20)
*added 1uL each of NEB2, Bam and Eco to each of the purified products:
**incubated at 37*C from 5:50-


==[[User:Conor McClune|Conor McClune]] 14:53, 25 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 21:15, 13 August 2010 (EDT)==
===iGEM10_020===
===Assembly of iGEM10_146 and iGEM10_149===
Bacterial lawns grew from both of the transformations from yesterday. Since there is no antibiotic for the correctly Eco/Bam/Bgl transfered product, I will have to do a number of colony PCRs to find one that took up the iGEM10_001 part.
====PCR magnification of Pcon part Bca1152====
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca9145
*recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
*run on 2K55
<br>
<br>
====Colony PCR====
'''Gel Verification'''
*oligos: ca56 (fwd) and igemTen22(rev)
*expected size:206
*picked 12 colonies from igem10_020 #15 and 4 colonies from igem10_020 #12
*gel purified the
''Gel Results''
The Expand seems to have worked best. It has a strong band at 200bp
*igem10_020 #15
 
[[image:ConorGel25.jpg|400px]]
====Digestions====
*igem10_020 #12
*for Bca1152 (Pcon)-recipe:
[[image:ConorGel26.jpg|400px]]<br>
**8uL of eluted PCR product
'''Results:''' no insertion in any of the chosen colonies
**1uL of NEB Buffer 2
*I may have forgotten to add DNTPs, which would explain why there are no bands
**1uL EcoRI
<br><br>
**1uL BamHI
I repicked 32 more colonies from igem10_020 #15 and ran a col PCR.
*for Bjh2252(lifeact gfp) and Bjh2251 (lifeact rfp):
''Gel Results''
** 14uL ddH2O
*igem10_020 #15 (expected band size: 1803)
** 2 uL NEB2+ATP Buffer
[[image:ConorGel27.jpg|400px]]
** 1uL EcoRI
*it appears each of the colonies have a plasmid that has iGEM10_001
** 1uL BamHI
** 1uL miniprepped plasmid
*incubated for 1hr at 37
*zymo cleaned FP digests (eluted with 20uL), small fragment zymo cleaned Bca1152 (
====Ligation====
*recipe:
**10uL vector digest (Bjh2251 or Bjh2251)
**7.5uL Bca1152
**2uL Ligase
**2uL T4 buffer +ATP
*let sit on counter for 30 min


===iGEM10_013===
====Transformation====
====Gibson Assembly====
*cell mixture: 100uL Jtk030, 15uL KCM
*DNA contents of assembly:
*50uL of cell mixture added to each ligation mixture
{| {{table}}
*let sit on ice for a few minutes
| align="center" style="background:#f0f0f0;"|'''Part'''
*heat shocked at 42*C for 90 sec
| align="center" style="background:#f0f0f0;"|'''Size'''
*added 100uL 2yt
| align="center" style="background:#f0f0f0;"|'''Band Brightness'''
*rescued for 45 min
| align="center" style="background:#f0f0f0;"|'''Weight'''
*plated both on CA
| align="center" style="background:#f0f0f0;"|'''Volume added (uL)'''
|-
| iGEM10_001||1335||bright||0.5||0.714285714
|-
| B10sbb10||1026||bright||0.5||0.714285714
|-
| iGEM10_009||279||dim||0.5||0.714285714
|-
| iGEM10_008||1057||bright||0.5||0.714285714
|-
| Bjh2294  w/ fwd oligo 19||2507||bright||0.5||0.714285714
|-
| p1601AC (vector)||3183||medium bright||1||1.428571429
|-
| ||||total||3.5||5
|}
*mixed with 15uL Gibson MM
*incubated at 50*C for 60 minutes
*transformed in to mc1061 cells


==[[User:Conor McClune|Conor McClune]] 14:25, 24 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 14:40, 12 August 2010 (EDT)==
===iGEM10_020===
===PCR magnification of Pcon part Bca1152===
====EcoR1/BamHI, EcoR1/Bglii Transfer====
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
'''Digestion'''
*recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
*the following wells were incubated at 37 for 30 min:
*run on 2K55
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Buffer'''
| align="center" style="background:#f0f0f0;"|'''Enzyme1'''
| align="center" style="background:#f0f0f0;"|'''Enzyme2'''
| align="center" style="background:#f0f0f0;"|'''Water'''
|-
| 1||4uL  iGem10_020 Gibson Colony #12 ||1uL NEB3||1uL Eco||1uL Bgl||4ul
|-
| 2||4uL  iGem10_020 Gibson Colony #15 ||1uL NEB3||1uL Eco||1uL Bgl||4ul
|-
| 3||8uL  iGem10_001 c||1uL NEB2||2uL Eco||2uL Bam||8ul
|}
<br>
<br>
I zymo purified the contents of the three wells, and spun it down into an equal volume of water as each digestion mixture (10,10,20uL)
'''Gel Verification'''
*expected size:1079


'''Ligation'''
[[image:ConorGel98.jpg|100px]]<br>
I incubated the following on my desk for 30 min:
The band looks too long
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part1'''
| align="center" style="background:#f0f0f0;"|'''Part2'''
| align="center" style="background:#f0f0f0;"|'''Buffer'''
| align="center" style="background:#f0f0f0;"|'''Enzyme1'''
| align="center" style="background:#f0f0f0;"|'''Water'''
|-
| 1||10uL  iGem10_020 Gibson Colony #12 ||10uL  iGem10_001 c||10uL Ligase Buffer||5uL T4 Ligase||65ul
|-
| 2||10uL  iGem10_020 Gibson Colony #15 ||10uL  iGem10_001 c||10uL Ligase Buffer||5uL T4 Ligase||65ul
|}


===iGEM10_007===
===Assembly of iGEM10_146 and iGEM10_149===
*sequencing company reported that oligo ca56 did not work with iGEM10_007, as it should have.
====Digestion====
*I ran a diagnostic PCR to see what was wrong:
*standard Eco/Bgl digestion of Bjh2252 (GFP) and Bjh2251 (RFP)
{| {{table}}
*11:50-1:00
| align="center" style="background:#f0f0f0;"|'''Well'''
*product was zymo purified
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''rev oligo'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
|-
| 1||iGEM10_007 #3||ca56||igemTen002||256
|-
| 2||iGEM10_007 #3||ca56||igemTen004||2044
|-
| 3||iGEM10_007 #3||igemTen003||igemTen008||1462
|-
| 4||iGEM10_007 #3||igemTen007||G00101||2635
|-
| 5||iGEM10_007 #7||ca56||igemTen002||256
|-
| 6||iGEM10_007 #7||ca56||igemTen004||2044
|-
| 7||iGEM10_007 #7||igemTen003||igemTen008||1462
|-
| 8||iGEM10_007 #7||igemTen007||G00101||2635
|}
''Gel Results''<br>
[[image:ConorGel23.jpg|400px]]


===iGEM10_013===
====Ligation====
*was able to continue parts preparation for Gibson assembly because my last primers arrived
*recipe: 3uL H20, 1uL T4 buffer, 1uL T4 ligase, 1uL FP part (Bjh2252 or Bjh2251), 4uL yesterdays Eco/Bam digest of Expand PCR of Bca1152
*incubated on desk from 11:50-12:30


'''Parts PCR'''<br>
====Transformation====
Well contents: 33uL polymerase,1uL each oligo, .5uL template DNA
*cell mixture: 200uL JTK030 cells, 30uL KCM
*run on program 2K45
*added 70uL of this mixture to each of the ligation mixtures
{| {{table}}
*heat shocked for 2 min
| align="center" style="background:#f0f0f0;"|'''Lane'''
*added 100ul 2YT
| align="center" style="background:#f0f0f0;"|'''Part'''
*rescued for 1.5 hours
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
*plated 100ul of iGEM10_146(lifact ffGFP) assembly on CA
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
*plated 100ul of iGEM10_149(lifact RFP) assembly on CA
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
|-
| 1|| B10sbb10||igemTen034||igemTen036||Expand||1050
|-
| 2||iGEM10_009||igemTen035||igemTen018||Expand||300
|-
| 3|| B10sbb10||igemTen034||igemTen036||Phusion||1050
|-
| 4||iGEM10_009||igemTen035||igemTen018||Phusion||300
|}
''''Gel Results''''<br>
[[image: ConorGel24.jpg|400px]]


==[[User:Conor McClune|Conor McClune]] 19:50, 23 June 2010 (EDT)==
===Picking colonies of iGEM10_145===
===Manual Assembly of iGEM10_004 and iGEM10_005===
*picked 4 green fluorescent colonies
*no growth on either of the plates, again
*ran a colony PCR with ca998 and G00101
*something must be wrong with ligation or digestion
**expected size: 964
*however, this was merely a backup for the Gibson Assembly of iGEM10_007, which seems to be working, so there is no need to troubleshoot
[[image:ConorGel99.jpg|200px]]
*no visible bands


===Gibson Assembly of iGEM10_007===
==[[User:Conor McClune|Conor McClune]] 17:30, 11 August 2010 (EDT)==
*sent miniprepped DNA in for sequencing
===PCR magnification of Pcon part Bca1152===
*I chose colony #3, because the restriction digest band was the brightest
*template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
*conducted PCR with both Phusion and Expand master mixes
*recipe: 50uL MM (phusion or expand), 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
*both run on 2K55
<br>
'''Gel Verification'''
*expected size:1079
*row 1 : 10uL phusion product
*row 2 : 10uL expand product


===Gibson Assembly of iGEM10_020===
[[image:ConorGel97.jpg|100px]]
*miniprepped colonies 1,3,4,11,12,14,15
*both products are the correct size, though yield is weak
*digested with Eco/Bam for restriction mapping
*Phusion product looks best
*Results:
[[image:ConorGel22.jpg|400px]]
Lane 8 (colony #15) is the only one that displays the correct bands


==[[User:Conor McClune|Conor McClune]] 15:34, 22 June 2010 (EDT)==
===Assembly of Lifact Payload Parts iGEM10_144-145===
===Manual Assembly of iGEM10_004 and iGEM10_005===
====Digestion====
*no growth on plates from yesterday, probably because I used only 1uL of ligation mixture for each transformation
*recipe: 4uL H20, 4uL DNA, 1uL NEB2, 1uL enzyme 1, 1uL enzyme 2
*luckily, I kept the ligation mixture, so I redid the transformations, using the remaining ligation mixture for each of the transformations
*digested Bjh1883 and jtk2541 with Eco/Bgl
*digested both phusion and expand PCR products of Bca1152 with Eco/Bam
*digested 4:53-


===Gibson Assembly of iGEM10_020===
====Zymo Cleanup====
*plenty of colonies on both plates
*conducted a standard zymo cleanup of Bjh1883 and jtk2541
*picked 8 colonies from each plate, and set up a colony PCR with oligos ca998 and igemTen22
*conducted a small-fragment zymo cleanup of PCR products
*expected magnified fragment length is 1617
====Transformation====
*colony PCR results:
*cell mixture: 200uL JTK030 cells, 30uL KCM
**plate 1(colonies 1-8):
*added 70uL of this mixture to each of the ligation mixtures
[[image:ConorGel19.jpg|400px]]
*heat shocked for 2 min
**plate 2(colonies 9-16):
*added 100ul 2YT
[[image:ConorGel20.jpg|400px]]
*rescued for 2.5 hours
*These colonies look good: 1,3,4,11,12,14,15
*plated 100ul of iGEM10_145(ffGFP) assembly on Spec
*plated 100ul of iGEM10_144(RFP) assembly on CA
**since we were out of CA plates, I diluted 25uL 1000X Amp with 75uL H20 and plated this onto a Chlor plate


===Gibson Assembly of iGEM10_007 (2nd Attempt)===
==[[User:Conor McClune|Conor McClune]] 15:07, 10 August 2010 (EDT)==
*miniprepped growth from yesterdays promising colonies (colonies 2,3,5,7,10,11,13,15)
===Testing the SLD in our transposase parts iGEM10_007,13,20===
**created 50uL DNA
Last night I picked colonies of each and grew them up in 6ml LB. iGEM10_007(piggybac) did not grow up because it was in the wrong color LB, but I repicked it at 11:30 and will test it later.
*Eco/Bam digested 4uL of each for restriction mapping
**mapping results (expected sizes are 7060 and 3200) :
[[image:ConorGel21.jpg|400px]]
*Results:
**7060 band is faint but definitely present in each lane
**3200band is probably present, but too faint to see, considering it is shorter
**these look good and ready for sequencing confirmation


==[[User:Conor McClune|Conor McClune]] 15:14, 21 June 2010 (EDT)==
iGEM10_013(sleeping beauty) and iGEM10_020(Tn5):
===Gibson Assembly iGEM10_007, Attempt #2===
*split each culture into two aliquots of 3mL - one with 3uL atc, one with nothing added
*cells grew up up on CA plate, no cells on negative control plates (K and Spec), besides the usual few tiny colonies on Spec
*placed these cultures on the incubator at 12:05
*picked 16 colonies:
**for 8 I did colony PCR using ca998 and G00101
**for 8 I did colony PCR with iGEM oligos 1 and 6
**growing up all 16 colonies in 1mL CA LB
====Colony PCR====
With primers ca998 and G00101 (colonies 1-8):
*expected band size: 9000
*gel picture:
[[image:ConorGel18.jpg|400px]]
*Results:  
**colonies 1,4,6,8 are too short
**colonies 2,3,5,7 show nothing, which is hopeful, since Taq generally cannot copy much larger than 5kb


With iGEM primers 1 and 6 (colonies 9-16):
results at 1:05: no lysis
*expected band size: 2186
*gel picture:
[[image:ConorGel17.jpg|400px]]
*Results:
**no strong bands
**numerous faint bands in lanes 2,3,5,7 (colonies 10,11,13,15) -- This suggests that something is being read off the primers
***These colonies are promising because the primers are from within the part


===Gibson Assembly of iGEM10_020===
results at 2:05: no lysis
*DNA contents:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''PCR Purified'''
| align="center" style="background:#f0f0f0;"|'''Size'''
| align="center" style="background:#f0f0f0;"|'''Band Brightness'''
| align="center" style="background:#f0f0f0;"|'''Weight'''
| align="center" style="background:#f0f0f0;"|'''Volume added (uL)'''
|-
| iGEM10_017||yes||1546||bright||0.5||0.833333333
|-
| iGEM10_016||yes||858||medium dim||1||1.666666667
|-
| Bjh2294 with fwd oligo 23||yes||2507||bright||0.5||0.833333333
|-
| p1601AC (vector)||yes||3183||medium bright||1||1.666666667
|-
| ||||||||||
|-
| ||||||total:||3||5
|}
*added 15uL Gibson MM
*incubated at 50*C for 70 minutes
*transformed the entire mixture into mc1061 cells
*plated cells on two CA LB plates incubated overnight


===Manual Assembly of iGEM10_004 and iGEM10_005===
==[[User:Conor McClune|Conor McClune]] 17:19, 9 August 2010 (EDT)==
'''Digestion'''
===Self lysis in Varied Media:Results===
*digested sbb04 and iGEM10_003 with lefty MM
Procedure:
*digested bjh2294 and iGEM10_002 with righty MM
*Grew up 10mL of two colonies off the plates for igem10_020, 013, and 131
*zymo cleaned all 4
*Used 8mL of those cells by filling 4 collection tubes with 2mL of cells. Spun them down and disposed of supernatant.
*ligated sbb04 with iGEM10_002 (to make iGEM10_004) and bjh2294 with iGEM10_003 (to make iGEM10_005)
*Resuspended one of each in TB, LB, Sea water, and Transposase Buffer.
*transformed iGEM10_005 into lefty and plated on KC
*Divided each of the 12 2mL sample into two wells on a 24 well plate.
*transformed iGEM10_004 into righty and plated on CA
*Added 1uL of atc to one of the 1mL samples from each 2mL sample. See detailed layout of plate above.
 
*Moved plate to 37deg shaker. Let sit for 1hour.
==[[User:Conor McClune|Conor McClune]] 19:16, 20 June 2010 (EDT)==
*Transfered 200uL of cells from each well to an eppendorph tube and spun down the cells. Transfered supernatant to Zymo Columns.
===Gibson Chemistry===
*Performed a zymo. Note: used 800ul of ADB to meet the necessary 1:4 ratio. Eluted with 10uL.
====Gibson Assembly of iGEM10_007 from June 18====
*Put tubes on ice. Transformed a huge batch of Righty cells (pir +). Added 70uL of cells to each tube.
*Tim picked 10 colonies yesterday and grew them up overnight
*Heat shocked, added 100uL of 2YT, let rescue for ~10min before realizing the plasmids are AC and I could just plate on Amp.
*miniprepped and digested with EcoRI and Xho1 for restriction mapping
*Plates are currently stored in incubator labeled 1-24 according to the layout above.
**expected fragment lengths: 9067 and 1196
[[Image:ConorGel16.jpg|400px]]
*none of these bands match the desired fragment lengths
*need to redo Gibson
====Gibson Assembly of iGEM10_007--Attempt #2====
*the 5uL DNA is made up of these volumes:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''PCR Purified'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_20 (atc)'''
| align="center" style="background:#f0f0f0;"|'''Size'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_20 (no atc)'''
| align="center" style="background:#f0f0f0;"|'''Band Brightness'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_013 (atc)'''
| align="center" style="background:#f0f0f0;"|'''Weight'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_013 (no atc)'''
| align="center" style="background:#f0f0f0;"|'''Volume added (uL)'''
| align="center" style="background:#f0f0f0;"|'''iGEM10_131 (atc)'''
|-
| align="center" style="background:#f0f0f0;"|'''iGEM10_131 (no atc)'''
| iGEM10_001||yes||1335|| bright||0.25||0.3125
|-
|-
| (B10sbb04)||yes||1788|| bright||0.25||0.3125
| Transposase Buffer||32||5||7||8||very high density||high density 
|-
|-
| iGEM10_002||yes||366||dim||0.5||0.625
| ASW||70||135||35||49||high density||higher density
|-
|-
| iGEM10_003||yes||1064||dim||1||1.25
| LB||490||146||119||105||high density (almost lawn)||high density (almost lawn)
|-
|-
| Self Lysis Device (Bjh2294)||yes||2507||medium bright||1||1.25
| TB||234||158||73||122||0||high density (almost lawn)
|-
| pBjh1601AC (vector)||yes||3183||medium bright||1||1.25
|-
| ||||||||||
|-
| ||||||total||4||5
|}
|}
*added 15uL Gibson MM
*incubated at 50*C for 60 min
*Heat shock tranformation(see general protocol) with these volumes:
**200uL competent mc1061
**30uL KCN
**20uL gibson DNA
**after rescue, I plated 70uL each on AC, K and Spec plates (K and Spec are negative controls)


==[[User:Conor McClune|Conor McClune]] 13:54, 18 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 14:31, 5 August 2010 (EDT)==
===Gibson Chemistry===
===Transposase Assay Results===
====Parts PCR====
No growth on any plates
'''RePCR of Bjh1601 AC on closest, black thermocycler'''
===His-tag addition to Transposases===
*Well contents: 33uL Phusion MM, 1uL each oligo (ss13r and igemTen009), .5uL template DNA
====PCR Isolation of Transposases====
*run on program 4K55
The following three pcr's were run on 2K55:
[[image:ConorGel14.jpg|400px]]
*This confirms that the problem I was having was due to the program I was using in the iGEM folder on the white thermocycler.
*This band looks perfect (3100bp) and is even stronger than the original.
*I gel purified it
<br>
<br>
'''DpnI Digestion of Bjh1601 AC'''<br>
*started with 8.5uL zymo-purified Bjh1601 AC from above procedure
*added 0.5uL DpnI and 0.9uL NEB buffer 2
*incubated for 1hour at 37*C
<br>
<br>
'''PCR of Bjh2294 with fwd oligos 19 and 23"<br>
Well Contents: 50uL phusion MM, 0.5uL DNA template, 1uL each oligo
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Name'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Vector'''
| align="center" style="background:#f0f0f0;"|'''Target Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Thermocycler Prgm'''
|-
|-
| 1||Bjh2294||Phusion||igemTen19||G00101||4K55
| 1||sbb10||KA||<SB100x!||ca998||igemTen048
|-
|-
| 2||Bjh2294||Phusion||igemTen23||G00101||4K55
| 2||sbb04||KA||<piggyBac!||ca998||igemTen050
|-
|-
| 3||Bjh2294||Phusion||igemTen19||G00101||4K45
| 3||iGEM10_055||KA||<tn5>{<NIS!}||ca998||igemTen051
|-
| 4||Bjh2294||Phusion||igemTen23||G00101||4K45
|}
|}


''Gel Results'' (the lane numbers correspond the the well numbers above)
==[[User:Conor McClune|Conor McClune]] 14:30, 4 August 2010 (EDT)==
<br>
===Transposase Assay: Testing Sleeping Beauty and Tn5 in NEB2+MgCl2, varying [DNA] and activity time===
[[image:ConorGel15.jpg|400px]]
 
*bands in lane 3 and 4 are strong and match the expected 2507bp
Transposition Buffer: Added 1.0165mg to each mL of NEB2  (MgCl2 tetrahydrate is 203.3g/mol and we're attempting to copy a transposition buffer that contains 150mM of MgCl2). We added 36.6mg of MgCl2 to 32.4mL of water and 3.6mL of 10xNEB2 to make '''150mM MgCl2 NEB2'''. We also added 5 drops of diluted HCl (tube labeled diluted HCl... we made it my dipping a pasteur pipette in the 12M HCL and then in 50mL of mgH20) to bring the pH from 7.8 to 7.6 or so.
**I gel purified these bands
 
Spun down all 5mLs of cells and 1uL of control cells (no ara). Resuspended in NEB2+MgCl2 buffer. Transferred control into a tube. Combined all 5mL resuspended cells into one tube, and then separated into 5 tubes.


====Gibson Assembly of iGEM10_007====
Added 1uL of atc and 10uL of arabinose to each mL of cells at 1:05pm. Put in 37deg shaker.
Well contents:
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Volume added (uL)'''
|-
| iGEM10_001||0.833
|-
| <piggybac!(B10sbb04)||0.833
|-
| iGEM10_002||0.833
|-
| iGEM10_003||0.833
|-
| Self Lysis Device (Bjh2294)||0.833
|-
| p1601AC (vector)||0.833
|-
| Gibson MM||20
|}


*Incubated for about 30 minutes at 50*C before somebody pulled it off the thermocycler
Started adding DNA at 1:40pm. For DNA, we mixed two minipreps of 9145-1144. Well, we mixed them after adding DNA from one of the MPs to the 1uL tubes and the 13 .5uL tube (Whoops).
*Incubated for an additional 30 minutes at 50*C
*Heat shock tranformation(see general protocol) with these volumes:
**200uL competent mc1061
**30uL KCN
**20uL gibson DNA
**plated 70uL each on AC, K and Spec plates (K and Spec are negative controls)


==[[User:Conor McClune|Conor McClune]] 13:54, 17 June 2010 (EDT)==
Somehow, we ran out of MP, so we're only adding up to 10uL of DNA.
===Gibson Chemistry===
====Parts Preparation====
'''DpnI Digestion''' <br>
I added 0.85 uL NEB2 buffer and 0.5uL DpnI to my Bjh1601 AC PCR product (gel purified) in order to digest all remaining template DNA, which would interfere with the Gibson reacton. The mixture was placed in the incubator at 10:26 and will be ready at 11:26.
*11.26: Zymo Preparation
**during zymo purification, I accidentally added 200 uL N3j Qiagen Neutralization buffer, instead of PE buffer.
**attempt to recover DNA:
***added 200uL PE to N3/DNA solution
***spun through zymo column in 30sec
***added 200 ADB buffer to solution
***spun through same zymo column in 30 sec
***discarded liquid waste
***spun 200uL PE though zymo column in 15 sec
***discarded liquid waste
***spun 200uL PE though zymo column in 15 sec
***discarded liquid waste
***spun for 90 sec to remove any remaining PE buffer
***discarded waste
***added 17uL H20
***spun into a new eppendorf in 30 seconds
***run on gel <br>
[[image:ConorGel11.jpg|400px]] <br>
I cut out the band at 3kb and gel purified it. The band is not intense, but it does seem I was able to recover some of the DNA. I gel purified this DNA, but I would prefer to use the product of the below PCR, if it is successful.


'''RePCR of Bjh1601 AC'''
Time points: <br>
*Well contents: 33uL Phusion MM, 1uL each oligo (ss13r and igemTen009), .5uL template DNA
.5hr- 2:10pm <br>
*run on program 4K55
1hr- 2:40pm <br>
[[image:ConorGel13.jpg|400px]]
1.5hr- 3:10pm <br>
*These were the exact procedures I used to originally PCR pBjh1601AC, with one difference: the thermocycler I used (I used the white one on the back wall and ran the 4k55 program under the iGEM folder. Note to future self: do not use programs in this folder). I will repeat this on the same thermocycler
2hr- 3:40pm


<br>
Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube.
'''PCR of sbb10'''
*Continued attempts to PCR sbb10
*double checked oligos on ApE
*created new 1:10 dilutions of oligos igemTen 14 and igemTen16
*in each well: 33uL polymerase MM, 1uL each of diluted igemTen 14 and igemTen16, 0.5 uL sbb10 from well A3 on Parts Plate 1
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
|-
| 1||sbb10||Phusion||igemTen14||igemTen16
|-
| 2||sbb11||Expand||igemTen14||igemTen16
|-
| 3||sbb12||Phusion||igemTen14||igemTen16
|-
| 4||sbb13||Expand||igemTen14||igemTen16
|}
Wells 1,2 run on program COL2K<br>
Wells 3,4 run on program 2K45 <br>
[[image:ConorGel12.jpg|400px]]
*still no bands appear in the desired 260bp range.  
*checked clotho --> the sequence we had entered for sbb10 on our "parts and data" spreadsheet was incorrect
*need to redesign primers igemTen14 and igemTen16
* I redesigned the primers


==[[User:Conor McClune|Conor McClune]] 13:52, 16 June 2010 (EDT)==
==[[User:Conor McClune|Conor McClune]] 16:53, 3 August 2010 (EDT)==
===Gibson Chemistry===
===Assembly of iGEM10_007===
====Parts PCR====
The assembly of this part is being put on pause until it is confirmed that the identical part iGEM10_131 is in fact incorrect. The plate with colonies is in the refrigerator.
''Gel Results from PCR of parts for assembly of iGEM10_013 and iGEM10_020''<br>
[[image:ConorGel9.jpg|400px]]


===Analytical PCR of iGEM10_131===
Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
*run on 4K45
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Size Expected'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
|-
| 1||B10sbb10||Expand||igemTen014||igemTen016||260||Very faint
| 1||iGEM10_103||ca56||igemTen004||2045||yes
|-
|-
| 2||iGEM10_009 A||Expand||igemTen015||igemTen018||310|| Yes
| 2||iGEM10_103||ca56||igemTen006||2411||yes
|-
|-
| 3||iGEM10_008d ||Expand||igemTen017||igemTen020||1090|| Yes
| 3||iGEM10_103||ca56||igemTen008||3475||no
|-
| 4||iGEM10_017 A||Expand||ca998||igemTen022||1650|| Yes
|-
| 5||iGEM10_016a||Expand||igemTen021||igemTen024||890|| Yes
|-
| 6||pB6 (BAC)||Expand||igemTen025||igemTen026||1000||Yes
|}
|}
*Gel Purified lanes 2-5 (iGEM10_009 A,iGEM10_008d,iGEM10_017 A and iGEM10_016a)
[[image:ConorGel96.jpg|400px]]
*the results of lane six demonstrate that our genomic miniprep of pB6 was successful and has a high concentration of BAC DNA


====PCR of B10sbb10, Bjh2294 and pBjh1601AC====
===Preparation of Bacterial Stock Choano Food===
*a matching rev backbone primer for pBjh1601AC was found: ss13r
*Grew up 400mL MC1061 overnight on 37*C shaker
*due to failure of previous pcr attempts for amplifying B10sbb10 and Bjh2294, I decided to try thermocycling at a lower temperature (45*C)
*split into two centrifuge bottles and centrifuged at 4200rpm for 14 minutes
*two well strips:
*poured off supernatant
**well contents: 33uL polymerase MM, 1uL each oligo, .5uL template DNA
*washed each pellet with 10mL artificial sea water (ASW)
**Strip 1: run on 4K45 (max temp=45*C)
*poured off ASW
{| {{table}}
*added 10mL ASW to each bottle and resuspended pellets
| align="center" style="background:#f0f0f0;"|'''Well'''
*centrifuged at 4200rpm for 14 minutes
| align="center" style="background:#f0f0f0;"|'''Part'''
*poured off supernatant
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
*resuspended pellets
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
*combined the contents of the two bottles
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
*created 2uL aliquots
|-
*
| 1||B10sbb10||Phusion||igemTen014||igemTen016
|-
| 2||B10sbb10||Expand||igemTen014||igemTen016
|-
| 3||Bjh2294||Phusion||igemTen007||G00101
|-
| 4||Bjh2294||Expand||igemTen007||G00101
|-
| 5||Bjh2294 (PCR Product)||Phusion||igemTen007||G00101
|}
**Strip 2: run on 4K55(max temp=55*C)
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Size Expected'''
|-
| 1||B10sbb10||Phusion||igemTen014||igemTen016||260
|-
| 2||pBjh1601||Phusion||igemTen009||ss13r||3183
|-
| 3||pBjh1601||Expand||igemTen009||ss13r||3183
|}


''Gel Results''
==[[User:Conor McClune|Conor McClune]] 14:39, 2 August 2010 (EDT)==
<br>
[[image:ConorGel10.jpg||400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Size Expected'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||B10sbb10||Phusion||igemTen014||igemTen016||260|| no
|-
| 2||B10sbb10||Expand||igemTen014||igemTen016||260|| no
|-
| 3||Bjh2294||Phusion||igemTen007||G00101||2540|| Probably just template DNA
|-
| 4||Bjh2294||Expand||igemTen007||G00101||2540||no
|-
| 5||Bjh2294 (PCR Product)||Phusion||igemTen007||G00101||2540|| yes
|-
| 6||-||-||-||-||-||
|-
| 7||B10sbb10||Phusion||igemTen014||igemTen016||260|| no
|-
| 8||pBjh1601||Phusion||igemTen009||ss13r||3183|| yes
|-
| 9||pBjh1601||Expand||igemTen009||ss13r||3183||no
|}
*none of the B10sbb10 PCR attempts worked
*I gel purified the desired bands in lanes 5 and 8:  Bjh2294 (PCR Product) and pBjh1601


==[[User:Conor McClune|Conor McClune]] 14:00, 15 June 2010 (EDT)==
===Miniprep of iGEM10_103===
===Gibson Chemistry===
The bacterial growth was smaller than usual, so I eluted with only 25uL H20, in order to avoid having a small concentration of DNA. I miniprepped all 6 picked clones of iGEM10_103.
====Parts PCR====
''Gel Results from yesterday's Expand PCR''<br>
*note: it appears some of well 6 (p1601ac) has evaporated. Perhaps the lid was not closed completely.
[[image: ConorGel6.jpg|400px]]
*gel looked messy, so I ran it again:
[[image: ConorGel7.jpg|400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_001 a||ca998||igemTen002||1360|| Yes
|-
| 3||iGEM10_001 b||ca998||igemTen002||1360||Yes
|-
| 5||iGEM10_001 c||ca998||igemTen002||1360||Yes
|-
| 7||<piggybac! (sbb04)||igemTen001||igemTen004||1820||Yes
|-
| 9||Self Lysis Device (Bjh2294)||igemTen007||G00101||2540||Very Faint
|-
| 11||1601AC plasmid||igemTen009||igemTen010||3220||No, not visible
|}
*Gel purified iGEM10_001-c (lane 5) and <piggybac!(sbb04) (lane 7)
*problem identified for PCR of p1601 AC : the reverse backbone oligo (igemTen10) does not match perfectly (it was originally designed for pMLL9-CA)


====RePCR of SLD and p1601 AC====
====Restriction Mapping====
{| {{table}}
*Digested 4uL of each miniprep with Xho/Bgl. Incubated from 11:30-12:15. Froze from 12:15-1:15.
| align="center" style="background:#f0f0f0;"|'''Well'''
*expected fragments: 6995 (desired fragment), 1607 (vector fragment)
| align="center" style="background:#f0f0f0;"|'''Part'''
[[image:ConorGel93.jpg|400px]]
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
*no bands visible
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
*I want to gel purify the larger of the two fragments, so I cut out the entire 7kb section of all six rows, hoping to capture even a low concentration of the correct piece.
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
|-
| 1||Self Lysis Device(Bjh2294)||Phusion||igemTen007||G00101
|-
| 2||Self Lysis Device(Bjh2294)||Expand||igemTen007||G00101
|-
| 3||Self Lysis Device(Bjh2294)||Taq||igemTen007||G00101
|-
| 4||1601AC plasmid||Phusion||igemTen009||G00101
|-
| 5||1601AC plasmid||Expand||igemTen009||G00101
|-
| 6||1601AC plasmid||Taq||igemTen009||G00101
|}
Well contents: 33uL polymerase MM, 1uL each oligo, 0.5uL template DNA
*Thermocycler program 4K55
[[image: ConorGel8.jpg|400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
|| 1||Self Lysis Device(Bjh2294)||Phusion||igemTen007||G00101||2540||Very Faint, but visible
|-
| 2||Self Lysis Device(Bjh2294)||Expand||igemTen007||G00101||2540||No, nothing visible
|-
| 3||Self Lysis Device(Bjh2294)||Taq||igemTen007||G00101||2540||Very Faint, but visible
|-
| 4||1601AC plasmid||Phusion||igemTen009||G00101||3220|| No, nothing visible
|-
| 5||1601AC plasmid||Expand||igemTen009||G00101||3220||No, nothing visible
|-
| 6||1601AC plasmid||Taq||igemTen009||G00101||3220||No, nothing visible
|}
*I gel purified the bands at 2540 in lanes 1 and 3 (both are Bjh2294, Self Lysis Device)


====PCR of  parts for assembly of iGEM10_013 and iGEM10_020====
Well contents: 33uL polymerase MM, 1uL each oligo, 0.5uL template DNA


====Analytical PCR====
Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
*run on 4K45
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Expected Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable Clones'''
|-
|-
| 1||B10sbb10||Expand||igemTen014||igemTen016
| 1||iGEM10_103||ca998||igemTen004||1859||1-3,5,6
|-
| 2||iGEM10_009 A||Expand||igemTen015||igemTen018
|-
| 3||iGEM10_008d ||Expand||igemTen017||igemTen020
|-
| 4||iGEM10_017 A||Expand||ca998||igemTen022
|-
|-
| 5||iGEM10_016a||Expand||igemTen021||igemTen024
| 2||iGEM10_103||ca998||igemTen006||2225||1-3,5,6
|-
|-
| 6||pB6 (BAC)||Expand||igemTen025||igemTen026
| 3||iGEM10_103||ca998||igemTen008||3289||none
|}
|}


==[[User:Conor McClune|Conor McClune]] 15:13, 14 June 2010 (EDT)==
'''Gel Pictures'''<Br>
===Gibson Assembly for iGEM10_007===
*ca998 + igemTen004
====Components====
[[image:ConorGel94.jpg|200px]]
*Parts (assembled in this order):
*ca998 + igemTen006 (lanes 1-6) and ca998 + igemTen008 (lanes 7-12)
**iGEM10_001
[[image:ConorGel95.jpg|200px]]
**<piggybac!> (part name B10sbb04)
**iGEM10_002
**iGEM10_003
**Self Lysis Device (part name Bjh2294)
*Vector:
**pBjh1601AC
====Parts PCR====
*I have already PCR-magnified iGEM10_002 and iGEM10_003 with the appropriate Gibson oligos I designed
''Procedure''<br> Well contents:
*50μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA


{| {{table}}
'''Results'''<br> Either a part is missing around the 3'TerminalRepeat, or the pcr is bad. I will repeat it tomorrow
| align="center" style="background:#f0f0f0;"|'''Wells'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
|-
| 1||iGEM10_001 a||ca998||igemTen002
|-
| 2||iGEM10_001 b||ca998||igemTen002
|-
| 3||iGEM10_001 c||ca998||igemTen002
|-
| 4||<piggybac!||igemTen001||igemTen004
|-
| 5||Self Lysis Device(Bjh2294)||igemTen007||G00101
|-
| 6||1601AC plasmid ||igemTen009||igemTen010
|}
*Run on PCR program 4K55 <br>
'''Gel Results''' <br>[[Image: ConorGel5.jpg||400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Acceptable?'''
|-
| 1||iGEM10_001 a||ca998||igemTen002||1360|| No, not visible
|-
| 3||iGEM10_001 b||ca998||igemTen002||1360||No, not visible
|-
| 5||iGEM10_001 c||ca998||igemTen002||1360||No, not visible
|-
| 7||<piggybac!||igemTen001||igemTen004||1820||No, not visible
|-
| 9||Self Lysis Device(Bjh2294)||igemTen007||G00101||2540||No, not visible
|-
| 11||1601AC plasmid||igemTen009||igemTen010||3220||No, not visible
|}
==== 2nd Attempt at PCR: Using Expand Polymerase====
*33μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Wells'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev oligo'''
|-
| 1||iGEM10_001 a||ca998||igemTen002
|-
| 2||iGEM10_001 b||ca998||igemTen002
|-
| 3||iGEM10_001 c||ca998||igemTen002
|-
| 4||<piggybac!||igemTen001||igemTen004
|-
| 5||Self Lysis Device(Bjh2294)||igemTen007||G00101
|-
| 6||1601AC plasmid ||igemTen009||igemTen010
|}
*Ran on 5K55 and left on thermocycler overnight (see gel results on tomorrow's notebook entry)
 
==[[User:Conor McClune|Conor McClune]] 14:11, 11 June 2010 (EDT)==
===Oligo and Composite Parts Test PCR #2 (continued from yesterday)===
15uL of PCR product run on gel:<br>
[[image:ConorGel2.jpg|400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Matches Gel Results?'''
|-
| 1||iGEM10_002d||igemTen003||G00101||500||yes
|-
| 2||iGEM10_002d||ca998||igemTen006||470||yes
|-
| 3||iGEM10_002d||ca998||G00101||570||yes
|-
| 4||iGEM10_003b||igemTen005||G00101||1190||yes
|-
| 5||iGEM10_003b||ca998||igemTen008||1160||yes
|-
| 6||iGEM10_003b||ca998||G00101||1260||yes
|-
| 7||iGEM10_003d||igemTen005||G00101||1190||yes
|-
| 8||iGEM10_003d||ca998||igemTen008||1160||yes
|-
| 9||iGEM10_003d||ca998||G00101||1260||yes
|}
<br>
<br>
 
===Oligo and Composite Parts Test PCR #3===
''Procedure''
Well contents:
*50μL polymerase MM, 1μL fwd oligo, 1μL rev oligo, 0.5μL DNA
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''well'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
|-
| 1||Taq||iGEM10_002d||igemTen003||igemTen006
|-
| 2||Phusion||iGEM10_002d||igemTen003||igemTen006
|-
| 3||Taq||iGEM10_003b||igemTen005||igemTen008
|-
| 4||Phusion||iGEM10_003b||igemTen005||igemTen008
|}
*Wells run on 2K55 on thermocycler.
''Gel Results''<br>
[[image:ConorGel3.jpg|400px]]
*possible DNA spilling from lane 4 to lanes 3&5, thus I repeated the gel run to avoid a false positive in lane 5 <br>
[[image:ConorGel4.jpg|400px]]
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Fwd Oligo'''
| align="center" style="background:#f0f0f0;"|'''Rev Oligo'''
| align="center" style="background:#f0f0f0;"|'''Predicted Size'''
| align="center" style="background:#f0f0f0;"|'''Matches Gel Results?'''
|-
| 1||Taq||iGEM10_002d||igemTen003||igemTen006||400||yes, strong band
|-
| 2||Phusion||iGEM10_002d||igemTen003||igemTen006||400||yes, weaker band
|-
| 3||Taq||iGEM10_003b||igemTen005||igemTen008||1100||yes, strong band
|-
| 4||Phusion||iGEM10_003b||igemTen005||igemTen008||1100||yes, weaker band
|-
| 5||n/a||pB6||||||40,000||possible band at 40kb, but very low conc.
|}
 
===Gel Purification of iGEM10_002 and iGEM10_003===
* cut out Phusion transcribed bands:
**band at 400 in lane 2
**band at 1100 in lane 4
*followed [http://openwetware.org/wiki/Template:SBB-Protocols_Zymo3 this protocol] and placed the purified PCR products of iGEM10_002 and iGEM10_003 in the working box
 
===PDD Part Preparation===
====Eco/Bam Transfers====
*Eco/Bam transfered 1601CA Bjh 2333 L, 1601CA Bjh2348 L,1601CA Bjh2349, and 1601CA Bjh 2331 in pBca1601 AK( we got this from shelley, and had to dilute it by half). We did this because BAC is CAM resistant, so if both parts are Cam resistant, it is possible to lose BAC, which is only single, and not be able to tell. So in the end, we will end up plating our stuff on A/K/C/Spec plates.
 
1601 AK or KA into MG1655 or MC1061
 
==[[User:Conor McClune|Conor McClune]] 13:23, 10 June 2010 (EDT)==
'''Negative Controls from Yesterday's Competent Cells'''
*plates: Amp,Kan,Cam,Spec
*positions: 1=pBca1256-Bjh2343, 2=pBca1256-Bjh2344, 3=pBca1256-Bjh1934, 4=MG1655
[[Image:NegControlAmp.jpg|200px]]
[[Image:NegControlKan.jpg|200px]]
[[Image:NegControlCam.jpg|200px]]
[[Image:NegControlSpec.jpg|200px]]<br> <br>
 
'''Plating yesterdays BAC tranformations failed'''<br>
[[Image:BAC2343.jpg|200px]]
[[Image:BAC2344&1934.jpg|200px]]<br>
''Retransformation''
* Transforming pB6 BAC into pBca1256-Bjh2343, pBca1256-Bjh2344 and pBca1256-Bjh1934
*adjustments to [http://openwetware.org/wiki/Template:SBB-Protocols_Micro1 procedure]:
**using only 15uL KCN for 98uL competent cells
**NOT diluting DNA by 10 before adding to cell solution
**For each strain, we split cell/KCN solution into two tubes of 57uL: we added 1μL DNA to one tube and 3 μL to the other tube
**after rescue, entire ~115uL of each solution was plated onto its own plate
<br> <br>
 
'''Oligo and Composite Parts Test PCR'''
Composite Parts ready to test:
*iGEM10_002d
**oligos:
***igemTen003
***igemTen006
*iGEM10_003b
**oligos:
***igemTen005
***igemTen008
*iGEM10_003d
**oligos:
***igemTen005
***igemTen008
Procedure:
*Dilute each oligo by ten (5ul oligo in 45ul MG H20)
*Combine in PCR tubes:
**50uL Phusion MM
**1uL oligo 1
**1uL oligo 2
**0.5uL miniprepped DNA
*program 2K55 started at 12:10 (should be done at 2:30)
Gel Results:
*no visible DNA in any of the lanes
[[Image:ConorGel1.jpg|200px]] <br> <br>
'''Oligo and Composite Parts Test PCR #2'''
*Parts tested:
**iGEM10_002d
***oligos:
****igemTen003(fwd)
****igemTen006(rev)
***iGEM10_003b
***oligos:
****igemTen005(fwd)
****igemTen008(rev)
***iGEM10_003d
***oligos:
****igemTen005(fwd)
****igemTen008(rev)
*for each part, testing:
**fwd part oligo + standard reverse plasmid oligo(G00101)
**rev part oligo + standard forward plasmid oligo(ca998)
**standard reverse plasmid oligo(G00101 + standard forward plasmid oligo(ca998)
 
Lanes:
#iGEM10_002d+igemTen003+G00101
#iGEM10_002d+ca998+igemTen006
#iGEM10_002d+ca998+G00101
#iGEM10_003b+igemTen005+G00101
#iGEM10_003b+ca998+igemTen008
#iGEM10_003b+ca998+G00101
#iGEM10_003d+igemTen005+G00101
#iGEM10_003d+ca998+igemTen008
#iGEM10_003d+ca998+G00101
 
PCR run and products kept at 16*C in the thermocycler overnight
 
==[[User:Conor McClune|Conor McClune]] 15:08, 9 June 2010 (EDT)==
'''Created -80 Stocks'''
*used [http://openwetware.org/wiki/-80_Glycerol_Stocks this protocol] to make -80 stocks for
**pBca1256-Bjh1934 in MG1655
**pB6 in DH5-alpha
**MG1655
**Bjh2343 in MG1655
**Bjh2344 in MG1655
*scanned and logged these stocks as the first iGEM -80 stocks (created a stock box for -80)
 
'''Made Competent Cells'''
*for pBca1256-Bjh1934 in MG1655,Bjh2343 in MG1655 and Bjh2344 in MG1655:
**added 500μL of overnight-grown culture to 50mL Spec LB in an Erlenmeyer flask
*for MG1655:
**added 1mL of overnight-grown culture to 50mL LB (no AB) in an Erlenmeyer flask (because the solution had been diluted 1:2 with 50% glycerol)
*both flasks put on shaker in lab at 12:00
 
*2:00pm
** pBca1256-Bjh1934,Bjh2343 and Bjh2344 (all in MG1655) have all grown up well, but not MG1655 (probably because of the glycerol)
*** MG1655 was place back in the incubator
*** pBca1256-Bjh1934,Bjh2343 and Bjh2344 solutions were chilled for ten minutes before being transferred to 50mL Falcon tubes and centrifuged for 13 minutes on 4100rpm.
****poured off the supernatant
****added 2.5ml TSS solution to each of the three vials
****made 98uL aliquots into 25 chilled eppendorf tubes for storage (25 tubes for each of pBca1256-Bjh1934,Bjh2343 and Bjh2344)
*Transformed BAC into 1, 2, 3.
*Plated on Cam, Spec plates.
 
*3:30
** removed MG1655  from incubator and conducted the procedure above (chilling, centrifuging, resuspending in TSS and aliquoting)
** 100 tubes labeled "1" (for Bjh2343), "2" ( Bjh2344), "3"(pBca1256-Bjh1934), "4"(MG1655 )
** Drops of the remaining solutions in the Falcon tubes were dropped on plates of Amp, Can, Spec, and Chlor as a negative control
 
==[[User:Conor McClune|Conor McClune]] 13:52, 8 June 2010 (EDT)==
*The five plates from yesterday (shown below) grew up well. We picked two colonies from the following:
**2343
**2344
**1256-1934
**MG1655
**pB6
*The first four of these will mixed with glycerol to be used as competent cells
*Picked Transformed cells from Stage 1 of Assembly of iGEM10_007
**plated on ACK plate to check for cotransformation
**incubated in shaker
**ran colony PCR
====Colony PCR====
Selected four colonies from each plate. Dipped the pipette tip in PCR tube with 40uL of MasterMix and a well containing 3mL of LB with the appropriate antibiotics, before spreading the pipette tip across a plate with all three antibiotics (to check for cotransformation).
 
iGEM10_001 (part 8 next to 23 in a CK vector): Expected band length: about 1500bp <br>
iGEM10_002 (part 11 next to 6 in AC vector): Expected band length: about 550bp <br>
iGEM10_003 (part 20 next to 5 in CK vector): Expected band length: about 1250bp <br>
 
[[Image:6-8-10.jpg]]
 
Colony PCR<br>
Lane 1 iGEM10_003a (should be 1250bp) <br>
Lane 2 iGEM10_003b (should be 1250bp) <br>
Lane 3 iGEM10_003c (should be 1250bp) <br>
4 iGEM10_003d (should be 1250bp) <br>
5 iGEM10_001a (should be 1500bp) <br>
6 iGEM10_001b (should be 1500bp)<br>
7 iGEM10_001c (should be 1500bp)<br>
8 iGEM10_001d (should be 1500bp)<br>
9 iGEM10_002a (should be 550bp)<br>
10 iGEM10_002b (should be 550bp)<br>
11 iGEM10_002c (should be 550bp)<br>
12 iGEM10_002d (should be 550bp)<br>
 
We will choose the following samples to miniprep tomorrow:<br>
iGEM10_001c,d <br>
iGEM10_002c,d <br>
iGEM10_003a,b <br>
 
*Moved the sequences from BioE 140L from Clotho to the Data Sheet on Google Docs
*Created oligos for a Gibson PCR for the assembly of iGEM10_007
**logged as the first oligos on Google Docs
**ordered by Tim
 
==[[User:Conor McClune|Conor McClune]] 14:38, 7 June 2010 (EDT)==
[[Image:jin_Notes1.JPG|200px]]<br>
*(with Tahoura)
*Transformed (spec)1256-2343 and (spec)1256-2344 into MG1655. Used minipreps of spec1256-2343 and spec1256-2344 from box and diluted 10x. Mixed 50ul MG1655 with 17.5ul KCN and added 1ul diluted DNA. Incubated for 1hr and plated on Spec. Left in incubator to grow <br>
*Plated pB6 (BAC), place in incubator. Needs to be picked and miniprepped tomorrow.<br>
*Transformed pBca1256-Bjh1934 into MG1655. Used procedure from (1)
*Attempted to enter parts into clotho --> would not save
 
===First step of Manual Assembly===


===Assembly of iGEM10_007===
====Digestion====
====Digestion====
Lefty MM added to 9+8, 7+11, 9+20<br>
*I used a stored, previously digested version of iGEM10_001
Right MM added to 7+23, 8+6, 7+5 <br>
*I used the above Xho/Bgl digestion of iGEM10_103 (aka iGEM10_006)
They should be done incubating at 3:48pm.<br>


====Ligation====
*Recipe: 7uL iGEM10_103 purified digest, 1uL iGEM10_001 purified digest, 1uL T4 Buffer, 1uL T4 ligase
*incubated on desk from 2:55-3:25


'''Lefty MasterMix''' (for 4uL miniprep DNA)
===Transformation===
*4uL water
*cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
*1uL of NEB2
*added 70uL of this cell mixture to the ligation mixture
*0.5uL XhoI
*heat shocked for 90sec
*0.5uL BamH1
*added 100uL 2YT and rescued for 1 hr
 
'''Righty MasterMix''' (for 4uL miniprep DNA)
*4uL water
*1uL of NEB2
*0.5uL XhoI
*0.5uL BglII


*note: righty digestions had different volumes, which seemed suspicious, so the digestion was repeated with 1uL of each enzyme for 30 min.
===Transformed iGEM10_013 #5===
*transformed into JTK030 cells for use in a transposase assay
*cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
*added 70uL of this cell mixture to the ligation mixture
*heat shocked for 90sec
*added 100uL 2YT and rescued for 1 hr


====Zymo====
===Grew up MC1061 cells to feed choanos in ASW===
Means of getting rid of restriction enzymes. Didn't heat kill because BamH1 isn't denatured by heat killing. Eluted with 10uL of H2O. Products are labeled 7+11 L, 9+8 L, 9+20 L.
4:00pm:
*added 100uL MC1061 competent cells to 400mL LB
*placed on shaker at 37*C overnight


====Ligation:====
==[[User:Conor McClune|Conor McClune]] 23:44, 1 August 2010 (EDT)==
Followed [http://openwetware.org/wiki/Template:SBB-Protocols_Enz4 this protocol] with the following amendments: <br>
===Picking colonies for iGEM10_103 (iGEM10_006)===
Mastermix
*Picked 6 colonies - grew up in KA LB
*6.5uL ddH2O
*Colony PCR
*1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
**primers: igemTen004 and ca56
*0.5uL T4 DNA Ligase
DNA
*3uL Lefty vector
*3uL Right vector
Added DNA to MM at 5:12pm. Will incubate on bench until 5:42pm.
 
===Transformation===
*70uL  of each ligation added to cells
**7+11/8+6 in righty
** 9+8/7+23 and 9+20/7+5 in lefty

Latest revision as of 00:19, 17 October 2010

June Entries
July Entries


To Do:

Conor McClune 22:47, 16 October 2010 (EDT)

Transposase Assembly

Colony PCR analysis

Colony PCR failed: no bands

  • 171+174 (A-D):
    • lanes 1-4
    • expected size: 4615
    • good clones:
  • 171+175 (A-B):
    • lanes 5-6
    • expected size: 4416
    • good clones:
  • 170+175 (A-C):
    • lanes 7-9
    • expected size: 5265
    • good clones:
  • 170+174 (A-B):
    • lanes 10-11
    • expected size: 5464
    • good clones:
  • 170+173 (A-D):
    • lanes 12-15
    • expected size: 5471
    • good clones:

Conor McClune 18:11, 13 October 2010 (EDT)

Transposase assembly

Conducted the following digestions:

Lane Part Restr. Enzymes Parent Size Expected Fragments Acceptable? Gel Purified
1 163-5a Xho/Bam 6004 4730, 1270 yes
2 163-5b Xho/Bam 6004 4730, 1270 yes yes
3 163-10a Xho/Bam 5150 3880, 1270 no
4 163-10b Xho/Bam 5150 3880, 1270 no
5 163-17b Xho/Bam 5400 4100, 1270 yes yes
6 163-17a Xho/Bam 5400 4100, 1270 yes
7 ladder
8 163-17b Xho/Bgl 5400 1475, 3921 yes
9 2142-164a Xho/Bgl 5090 3408, 1680 yes yes
10 2142-164b Xho/Bgl 5090 3408, 1680 yes
11 2142-165a Xho/Bgl 5080 3401, 1680 yes yes
12 2142-165b Xho/Bgl 5080 3401, 1680 yes
13 2142-166a Xho/Bgl 4880 3200, 1680 yes
14 2142-166b Xho/Bgl 4880 3200, 1680 yes yes

Gel image:

preparation for plasmid payload cytometry

Self lysis test

  • started 2:00
  • checked at 4:30:
1 2 3 6 7
51 lysed lysed lysed barely lysed lysed
51 lysed lysed lysed lysed lysed


preparation of cells

  • scraped choanos and put 1ml in each test tube
  • added 40 uL bacteria to each test tube at 2:30
  • induced with arabinose fifteen minutes later (at 2:45)

Conor McClune 18:31, 12 October 2010 (EDT)

  • flow cytometry got no hits, because the choanos were in ASW, so the bacteria did not lyse

Conor McClune 18:31, 11 October 2010 (EDT)

preparation for plasmid payload cytometry

Self lysis test

  • started 2:15
  • checked at 4:15:
1 2 3 6 7
51 lysed lysed lysed lysed lysed
51 lysed lysed lysed lysed lysed


preparation of cells

  • scraped choanos and put 6ml in each test tube
  • added 240 uL bacteria to each test tube
  • induced with arabinose fifteen minutes later (at 2:30)

Conor McClune 19:02, 6 October 2010 (EDT)

Plasmid Delivery Assay

Plasmid Payload Numbers:

Number Part Antibiotic
1 pDYAL eGFP Amp
2 pDYAL eGFP Swar Amp
3 Clipit GFP Amp
4 M cherry Amp
5 Tera\'s α-tub GFP jtk159 Amp
6 Tera\'s efla GFP jtk160 Amp
7 PfuGWP GFP Amp
8 Tera\'s myo GFP jtk162 Amp
  • 51 and 52 refer to the Self-lysis/Vacuole-buster devices iGEM10_051-052

Procedure

Choanos

  • aspirated two plates (15ml each) of choanos in ASW, resuspended each in 5mL of choano media
    • note: the choano media we planned on using was contaminated, so I had to use an older one (one that seemed to be lacking something because choanos have not grown very well in it)
  • scraped choanos, mixed the two plates (to get 10mL), aliquoted 500uL into each of 12 mini slide wells
  • added 20uL of bacteria to the wells (10:15am):
1+51 2+51 3+51 6+51 7+51 lifeact iGEM10_171 +51(color control)
1+52 2+52 3+52 6+52 7+52 white bacteria
  • mistake:added 0.5uL ATC to each well at 10:30am, instead of arabinose (SLD is under Pbad not Ptet)
  • correction: added 5uL arabinose to each well at 11:00am

Self lysis test

    • made two 2mL aliquots of each of the plasmid bacteria cultures listed above
    • added 2uL ATC (mistake) and 20uL of arabinose to one of each of the aliquots of each culture

Results

Self Lysis Test

  • after 5 hours, all 10 plasmid+SLD/VB cultures with arabinose had lysed.

Conor McClune 22:30, 26 September 2010 (EDT)

Eco/Bam transfer of iGEM10_171 (A) into Bca1256

  • transformed product into JTK159 and plated on spec

Re-Assembly of iGEM10_170

  • gel purified my previous digestions of iGEM10_159 and Bjh1906
  • ligated 4uL of each with 1uL t4 buffer and 1uL t4 ligase
  • transformed into JTK159 and plated on CA

Conor McClune 16:34, 23 September 2010 (EDT)

LifeAct Assembly

Of the colonies if iGEM10_170 and iGEM10_171 picked yesterday, colonies 5,8,12 and 15 were cotransformed

Miniprepped: Colonies 1,2,3,7 (iGEM10_170) and colonies 10,11,18, 20 (iGEM10_171) because they were the reddest

  • renamed these clones iGEM10_170 A-D and iGEM10_171 A-D, respectively
    • iGEM10_171A sequenced well, with one mutation in the promoter
    • all of iGEM10_170 A-D are missing the terminator

Conor McClune 19:55, 22 September 2010 (EDT)

LifeAct Assembly

There is growth on the plates I made yesterday for iGEM10_170-171, though the Red/green colonies on iGEM10_171 are very small

I picked 8 colonies for iGEM10_170 and 12 from iGEM10_171 (only the last 4 may be green AND red).

Ran a colony PCR, but it did not show much because the addition of the terminator is not visible on the gel. The gel for the last colonies

  • lanes 1-8: iGEM10_170
    • all but colony 8 look good
  • lanes 9-13: iGEM10_171
    • all look good, except that col PCR failed for colony 12

Conor McClune 18:18, 21 September 2010 (EDT)

Restriction digest (Eco/Bam)of iGEM10_159-160

  • used iGEM10_159C
  • expected band sizes: 2660, 1580

  • the bands look good for both (faintess can be attributed to a bad zymo-forgot to spin out solvent for 90 sec before adding water)

Assembly of iGEM

Digested:

  • Xho/Bam: iGEM10_159C and iGEM10_160
  • Xho/Bgl: Bjh1906

Ligated each of the iGEM parts to left of Bjh1906 to make iGEM10_170-171, respectively
Transformed into JTK159 cells and plated on CA

Conor McClune 19:59, 18 September 2010 (EDT)

LifeAct Assembly

Redigestion of iGEM10_156A and iGEM10_159D

(this is necessary because the previous version of

  • Both were digested with Xho/Bam for 30 min
  • Zymo'd
  • Ligation (5:00-5:30):
    • iGEM10_156A + Bjh1881
    • iGEM10_159D + Bjh1906 (terminator)
    • iGEM10_159A + Bjh1906 (terminator)

Results: no growth for the iGEM10_159 + Bjh1906 assemblies, but one red/green colony for iGEM10_156A + Bjh1881 = iGEM10_160

  • colony PCR of iGEM10_160:

  • expected band : 1800bp, the band looks good

Remaking iGEM10_161 and iGEM10_162

(This is necessary because I had mislabelled iGEM10_157-158)
yesterday:

  • ligated the predigested combinations:
    • iGEM10_157 + Bjh1881
    • iGEM10_158 + Jtk2541

today:

  • transformed both ligations into JTK159 strain
  • plated on CK

Results: no growth, failed assembly

Conor McClune 18:38, 17 September 2010 (EDT)

LifeAct Assembly

Restriction digest of iGEM10_159-162 with Eco/Bam

  • expected sizes for all bands: 2660, 1580
  • iGEM10_159: lanes 1-4
  • iGEM10_160: lanes 5-8
  • iGEM10_161: lanes 9-12
  • iGEM10_162: lanes 13-16

Conor McClune 18:38, 14 September 2010 (EDT)

Assembly of LifeAct Payload

Potential Error discovered: plate with Eco/Bam iGEM157 and iGEM10158 were probably mislabelled since 157 was red and 158 was green (they should have been the green and red, respectively). This means that iGEM161-162 are probably just two copies of the same FP, one lifeact and one not.

Conor McClune 22:03, 12 September 2010 (EDT)

Lifeact Payload assembly

Digestion

  • Lefty digested: iGEM10_155-158
  • Righty digested: Bjh1881, Jtk2541
  • incubated 3:30-4:20

Ligation

  • ligating the following combinations:
    • iGEM10_155 + Jtk2541
    • iGEM10_156 + Bjh1881
    • iGEM10_157 + Bjh1881
    • iGEM10_158 + Jtk2541

Conor McClune 01:09, 10 September 2010 (EDT)

Lifact Assembly

  • Eco/Bam'd iGEM10_155-158 into pMLL9 (CA) (for continued assembly) and also into 1256 (Spec) (for assaying in choanos)

Conor McClune 01:28, 9 September 2010 (EDT)

Lifact Payload Assembly

  • I Eco/Bam digested iGEM10_155-158
  • Gel purified smaller fragment of each
  • meant to eco/bam into pMLL CA but we were out of the digested vector. I will have to digest this tomorrow

Conor McClune 18:04, 7 September 2010 (EDT)

Lifact Assembly

Colony PCR results

  • colony # corresponds to lane #
  • expected sizes:
    • lanes 1-4 Bca1237 + Bjh2252:1070
    • lanes 5-8 Bca1238 + Bjh2252:1070
    • lanes 9-4 Bca1237 + Bjh2251:1020
    • lanes 1-4 Bca1238 + Bjh2251:1020

  • note: broken well in lane 8, so contents were put in adjacent well

Results:

  • all lanes look good

Conor McClune 21:06, 5 September 2010 (EDT)

Lifact Payload Assembly

  • Ran a gel of Tahoura's PCR of Pcon parts Bca1237 and Bca1238:
    • image:
    • cut out 200-300bp range for both parts and small fragment gel purified (eluted with 8.5uL H20)
  • added 1uL each of NEB2, Bam and Eco to each of the purified products:
    • incubated at 37*C from 5:50-

Conor McClune 21:15, 13 August 2010 (EDT)

Assembly of iGEM10_146 and iGEM10_149

PCR magnification of Pcon part Bca1152

  • template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca9145
  • recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
  • run on 2K55


Gel Verification

  • expected size:206
  • gel purified the

The Expand seems to have worked best. It has a strong band at 200bp

Digestions

  • for Bca1152 (Pcon)-recipe:
    • 8uL of eluted PCR product
    • 1uL of NEB Buffer 2
    • 1uL EcoRI
    • 1uL BamHI
  • for Bjh2252(lifeact gfp) and Bjh2251 (lifeact rfp):
    • 14uL ddH2O
    • 2 uL NEB2+ATP Buffer
    • 1uL EcoRI
    • 1uL BamHI
    • 1uL miniprepped plasmid
  • incubated for 1hr at 37
  • zymo cleaned FP digests (eluted with 20uL), small fragment zymo cleaned Bca1152 (

Ligation

  • recipe:
    • 10uL vector digest (Bjh2251 or Bjh2251)
    • 7.5uL Bca1152
    • 2uL Ligase
    • 2uL T4 buffer +ATP
  • let sit on counter for 30 min

Transformation

  • cell mixture: 100uL Jtk030, 15uL KCM
  • 50uL of cell mixture added to each ligation mixture
  • let sit on ice for a few minutes
  • heat shocked at 42*C for 90 sec
  • added 100uL 2yt
  • rescued for 45 min
  • plated both on CA

Conor McClune 14:40, 12 August 2010 (EDT)

PCR magnification of Pcon part Bca1152

  • template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
  • recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
  • run on 2K55


Gel Verification

  • expected size:1079


The band looks too long

Assembly of iGEM10_146 and iGEM10_149

Digestion

  • standard Eco/Bgl digestion of Bjh2252 (GFP) and Bjh2251 (RFP)
  • 11:50-1:00
  • product was zymo purified

Ligation

  • recipe: 3uL H20, 1uL T4 buffer, 1uL T4 ligase, 1uL FP part (Bjh2252 or Bjh2251), 4uL yesterdays Eco/Bam digest of Expand PCR of Bca1152
  • incubated on desk from 11:50-12:30

Transformation

  • cell mixture: 200uL JTK030 cells, 30uL KCM
  • added 70uL of this mixture to each of the ligation mixtures
  • heat shocked for 2 min
  • added 100ul 2YT
  • rescued for 1.5 hours
  • plated 100ul of iGEM10_146(lifact ffGFP) assembly on CA
  • plated 100ul of iGEM10_149(lifact RFP) assembly on CA

Picking colonies of iGEM10_145

  • picked 4 green fluorescent colonies
  • ran a colony PCR with ca998 and G00101
    • expected size: 964

  • no visible bands

Conor McClune 17:30, 11 August 2010 (EDT)

PCR magnification of Pcon part Bca1152

  • template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
  • conducted PCR with both Phusion and Expand master mixes
  • recipe: 50uL MM (phusion or expand), 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
  • both run on 2K55


Gel Verification

  • expected size:1079
  • row 1 : 10uL phusion product
  • row 2 : 10uL expand product

  • both products are the correct size, though yield is weak
  • Phusion product looks best

Assembly of Lifact Payload Parts iGEM10_144-145

Digestion

  • recipe: 4uL H20, 4uL DNA, 1uL NEB2, 1uL enzyme 1, 1uL enzyme 2
  • digested Bjh1883 and jtk2541 with Eco/Bgl
  • digested both phusion and expand PCR products of Bca1152 with Eco/Bam
  • digested 4:53-

Zymo Cleanup

  • conducted a standard zymo cleanup of Bjh1883 and jtk2541
  • conducted a small-fragment zymo cleanup of PCR products

Transformation

  • cell mixture: 200uL JTK030 cells, 30uL KCM
  • added 70uL of this mixture to each of the ligation mixtures
  • heat shocked for 2 min
  • added 100ul 2YT
  • rescued for 2.5 hours
  • plated 100ul of iGEM10_145(ffGFP) assembly on Spec
  • plated 100ul of iGEM10_144(RFP) assembly on CA
    • since we were out of CA plates, I diluted 25uL 1000X Amp with 75uL H20 and plated this onto a Chlor plate

Conor McClune 15:07, 10 August 2010 (EDT)

Testing the SLD in our transposase parts iGEM10_007,13,20

Last night I picked colonies of each and grew them up in 6ml LB. iGEM10_007(piggybac) did not grow up because it was in the wrong color LB, but I repicked it at 11:30 and will test it later.

iGEM10_013(sleeping beauty) and iGEM10_020(Tn5):

  • split each culture into two aliquots of 3mL - one with 3uL atc, one with nothing added
  • placed these cultures on the incubator at 12:05

results at 1:05: no lysis

results at 2:05: no lysis

Conor McClune 17:19, 9 August 2010 (EDT)

Self lysis in Varied Media:Results

Procedure:

  • Grew up 10mL of two colonies off the plates for igem10_020, 013, and 131
  • Used 8mL of those cells by filling 4 collection tubes with 2mL of cells. Spun them down and disposed of supernatant.
  • Resuspended one of each in TB, LB, Sea water, and Transposase Buffer.
  • Divided each of the 12 2mL sample into two wells on a 24 well plate.
  • Added 1uL of atc to one of the 1mL samples from each 2mL sample. See detailed layout of plate above.
  • Moved plate to 37deg shaker. Let sit for 1hour.
  • Transfered 200uL of cells from each well to an eppendorph tube and spun down the cells. Transfered supernatant to Zymo Columns.
  • Performed a zymo. Note: used 800ul of ADB to meet the necessary 1:4 ratio. Eluted with 10uL.
  • Put tubes on ice. Transformed a huge batch of Righty cells (pir +). Added 70uL of cells to each tube.
  • Heat shocked, added 100uL of 2YT, let rescue for ~10min before realizing the plasmids are AC and I could just plate on Amp.
  • Plates are currently stored in incubator labeled 1-24 according to the layout above.
' iGEM10_20 (atc) iGEM10_20 (no atc) iGEM10_013 (atc) iGEM10_013 (no atc) iGEM10_131 (atc) iGEM10_131 (no atc)
Transposase Buffer 32 5 7 8 very high density high density
ASW 70 135 35 49 high density higher density
LB 490 146 119 105 high density (almost lawn) high density (almost lawn)
TB 234 158 73 122 0 high density (almost lawn)

Conor McClune 14:31, 5 August 2010 (EDT)

Transposase Assay Results

No growth on any plates

His-tag addition to Transposases

PCR Isolation of Transposases

The following three pcr's were run on 2K55:

Well Name Vector Target Part Fwd Oligo Rev Oligo
1 sbb10 KA <SB100x! ca998 igemTen048
2 sbb04 KA <piggyBac! ca998 igemTen050
3 iGEM10_055 KA <tn5>{<NIS!} ca998 igemTen051

Conor McClune 14:30, 4 August 2010 (EDT)

Transposase Assay: Testing Sleeping Beauty and Tn5 in NEB2+MgCl2, varying [DNA] and activity time

Transposition Buffer: Added 1.0165mg to each mL of NEB2 (MgCl2 tetrahydrate is 203.3g/mol and we're attempting to copy a transposition buffer that contains 150mM of MgCl2). We added 36.6mg of MgCl2 to 32.4mL of water and 3.6mL of 10xNEB2 to make 150mM MgCl2 NEB2. We also added 5 drops of diluted HCl (tube labeled diluted HCl... we made it my dipping a pasteur pipette in the 12M HCL and then in 50mL of mgH20) to bring the pH from 7.8 to 7.6 or so.

Spun down all 5mLs of cells and 1uL of control cells (no ara). Resuspended in NEB2+MgCl2 buffer. Transferred control into a tube. Combined all 5mL resuspended cells into one tube, and then separated into 5 tubes.

Added 1uL of atc and 10uL of arabinose to each mL of cells at 1:05pm. Put in 37deg shaker.

Started adding DNA at 1:40pm. For DNA, we mixed two minipreps of 9145-1144. Well, we mixed them after adding DNA from one of the MPs to the 1uL tubes and the 13 .5uL tube (Whoops).

Somehow, we ran out of MP, so we're only adding up to 10uL of DNA.

Time points:
.5hr- 2:10pm
1hr- 2:40pm
1.5hr- 3:10pm
2hr- 3:40pm

Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube.

Conor McClune 16:53, 3 August 2010 (EDT)

Assembly of iGEM10_007

The assembly of this part is being put on pause until it is confirmed that the identical part iGEM10_131 is in fact incorrect. The plate with colonies is in the refrigerator.

Analytical PCR of iGEM10_131

Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA

  • run on 4K45
Well Part Fwd Oligo Rev Oligo Expected Size Acceptable?
1 iGEM10_103 ca56 igemTen004 2045 yes
2 iGEM10_103 ca56 igemTen006 2411 yes
3 iGEM10_103 ca56 igemTen008 3475 no

Preparation of Bacterial Stock Choano Food

  • Grew up 400mL MC1061 overnight on 37*C shaker
  • split into two centrifuge bottles and centrifuged at 4200rpm for 14 minutes
  • poured off supernatant
  • washed each pellet with 10mL artificial sea water (ASW)
  • poured off ASW
  • added 10mL ASW to each bottle and resuspended pellets
  • centrifuged at 4200rpm for 14 minutes
  • poured off supernatant
  • resuspended pellets
  • combined the contents of the two bottles
  • created 2uL aliquots

Conor McClune 14:39, 2 August 2010 (EDT)

Miniprep of iGEM10_103

The bacterial growth was smaller than usual, so I eluted with only 25uL H20, in order to avoid having a small concentration of DNA. I miniprepped all 6 picked clones of iGEM10_103.

Restriction Mapping

  • Digested 4uL of each miniprep with Xho/Bgl. Incubated from 11:30-12:15. Froze from 12:15-1:15.
  • expected fragments: 6995 (desired fragment), 1607 (vector fragment)

  • no bands visible
  • I want to gel purify the larger of the two fragments, so I cut out the entire 7kb section of all six rows, hoping to capture even a low concentration of the correct piece.


Analytical PCR

Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA

  • run on 4K45
Well Part Fwd Oligo Rev Oligo Expected Size Acceptable Clones
1 iGEM10_103 ca998 igemTen004 1859 1-3,5,6
2 iGEM10_103 ca998 igemTen006 2225 1-3,5,6
3 iGEM10_103 ca998 igemTen008 3289 none

Gel Pictures

  • ca998 + igemTen004

  • ca998 + igemTen006 (lanes 1-6) and ca998 + igemTen008 (lanes 7-12)

Results
Either a part is missing around the 3'TerminalRepeat, or the pcr is bad. I will repeat it tomorrow

Assembly of iGEM10_007

Digestion

  • I used a stored, previously digested version of iGEM10_001
  • I used the above Xho/Bgl digestion of iGEM10_103 (aka iGEM10_006)

Ligation

  • Recipe: 7uL iGEM10_103 purified digest, 1uL iGEM10_001 purified digest, 1uL T4 Buffer, 1uL T4 ligase
  • incubated on desk from 2:55-3:25

Transformation

  • cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
  • added 70uL of this cell mixture to the ligation mixture
  • heat shocked for 90sec
  • added 100uL 2YT and rescued for 1 hr

Transformed iGEM10_013 #5

  • transformed into JTK030 cells for use in a transposase assay
  • cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
  • added 70uL of this cell mixture to the ligation mixture
  • heat shocked for 90sec
  • added 100uL 2YT and rescued for 1 hr

Grew up MC1061 cells to feed choanos in ASW

4:00pm:

  • added 100uL MC1061 competent cells to 400mL LB
  • placed on shaker at 37*C overnight

Conor McClune 23:44, 1 August 2010 (EDT)

Picking colonies for iGEM10_103 (iGEM10_006)

  • Picked 6 colonies - grew up in KA LB
  • Colony PCR
    • primers: igemTen004 and ca56
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